Sloth biology: an update on their physiological ecology, behavior and role as vectors of arthropods and arboviruses

This is a review of the research undertaken since 1971 on the behavior and physiological ecology of sloths. The animals exhibit numerous fascinating features. Sloth hair is extremely specialized for a wet tropical environment and contains symbiotic algae. Activity shows circadian and seasonal variation. Nutrients derived from the food, particularly in Bradypus, only barely match the requirements for energy expenditure. Sloths are hosts to a fascinating array of commensal and parasitic arthropods and are carriers of various arthropod-borne viruses. Sloths are known reservoirs of the flagellate protozoan which causes leishmaniasis in humans, and may also carry trypanosomes and the protozoan Pneumocystis carinii.

Mathematical modeling and optimal control of malaria

Malaria continues to infect millions and kill hundreds of thousands of people worldwide each year, despite over a century of research and attempts to control and eliminate this infectious disease. Challenges such as the development and spread of drug resistant malaria parasites, insecticide resistance to mosquitoes, climate change, the presence of individuals with subpatent malaria infections which normally are asymptomatic and behavioral plasticity in the mosquito hinder the prospects of malaria control and elimination. In this thesis, mathematical models of malaria transmission and control that address the role of drug resistance, immunity, iron supplementation and anemia, immigration and visitation, and the presence of asymptomatic carriers in malaria transmission are developed. A within-host mathematical model of severe Plasmodium falciparum malaria is also developed. First, a deterministic mathematical model for transmission of antimalarial drug resistance parasites with superinfection is developed and analyzed. The possibility of increase in the risk of superinfection due to iron supplementation and fortification in malaria endemic areas is discussed. The model results calls upon stakeholders to weigh the pros and cons of iron supplementation to individuals living in malaria endemic regions. Second, a deterministic model of transmission of drug resistant malaria parasites, including the inflow of infective immigrants, is presented and analyzed. The optimal control theory is applied to this model to study the impact of various malaria and vector control strategies, such as screening of immigrants, treatment of drug-sensitive infections, treatment of drug-resistant infections, and the use of insecticide-treated bed nets and indoor spraying of mosquitoes. The results of the model emphasize the importance of using a combination of all four controls tools for effective malaria intervention. Next, a two-age-class mathematical model for malaria transmission with asymptomatic carriers is developed and analyzed. In development of this model, four possible control measures are analyzed: the use of long-lasting treated mosquito nets, indoor residual spraying, screening and treatment of symptomatic, and screening and treatment of asymptomatic individuals. The numerical results show that a disease-free equilibrium can be attained if all four control measures are used. A common pitfall for most epidemiological models is the absence of real data; model-based conclusions have to be drawn based on uncertain parameter values. In this thesis, an approach to study the robustness of optimal control solutions under such parameter uncertainty is presented. Numerical analysis of the optimal control problem in the presence of parameter uncertainty demonstrate the robustness of the optimal control approach that: when a comprehensive control strategy is used the main conclusions of the optimal control remain unchanged, even if inevitable variability remains in the control profiles. The results provide a promising framework for the design of cost-effective strategies for disease control with multiple interventions, even under considerable uncertainty of model parameters. Finally, a separate work modeling the within-host Plasmodium falciparum infection in humans is presented. The developed model allows re-infection of already-infected red blood cells. The model hypothesizes that in severe malaria due to parasite quest for survival and rapid multiplication, the Plasmodium falciparum can be absorbed in the already-infected red blood cells which accelerates the rupture rate and consequently cause anemia. Analysis of the model and parameter identifiability using Markov chain Monte Carlo methods is presented.

Molecular identification of Sicilian (dß)º-thalassemia associated with ß-thalassemia and hemoglobin S in Brazil

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (dß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (dß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(dß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (dß)º-thalassemia in association with ß-thalassemia documented at the molecular level.

Spectroscopic Studies of III-V Semiconductor Materials for Improved Devices

Defects in semiconductor crystals and at their interfaces usually impair the properties and the performance of devices. These defects include, for example, vacancies (i.e., missing crystal atoms), interstitials (i.e., extra atoms between the host crystal sites), and impurities such as oxygen atoms. The defects can decrease (i) the rate of the radiative electron transition from the conduction band to the valence band, (ii) the amount of charge carriers, and (iii) the mobility of the electrons in the conduction band. It is a common situation that the presence of crystal defects can be readily concluded as a decrease in the luminescence intensity or in the current flow for example. However, the identification of the harmful defects is not straightforward at all because it is challenging to characterize local defects with atomic resolution and identification. Such atomic-scale knowledge is however essential to find methods for reducing the amount of defects in energy-efficient semiconductor devices. The defects formed in thin interface layers of semiconductors are particularly difficult to characterize due to their buried and amorphous structures. Characterization methods which are sensitive to defects often require well-defined samples with long range order. Photoelectron spectroscopy (PES) combined with photoluminescence (PL) or electrical measurements is a potential approach to elucidate the structure and defects of the interface. It is essential to combine the PES with complementary measurements of similar samples to relate the PES changes to changes in the interface defect density. Understanding of the nature of defects related to III-V materials is relevant to developing for example field-effect transistors which include a III-V channel, but research is still far from complete. In this thesis, PES measurements are utilized in studies of various III-V compound semiconductor materials. PES is combined with photoluminescence measurements to study the SiO2/GaAs, SiNx/GaAs and BaO/GaAs interfaces. Also the formation of novel materials InN and photoluminescent GaAs nanoparticles are studied. Finally, the formation of Ga interstitial defects in GaAsN is elucidated by combining calculational results with PES measurements.

New [18F]Tracers for Alzheimer's Disease Imaging. Labeling Synthesis And Biological Testing

Alzheimer’s disease (AD) is the most common form of dementia. Characteristic changes in an AD brain are the formation of β-amyloid protein (Aβ) plaques and neurofibrillary tangles, though other alterations in the brain have also been connected to AD. No cure is available for AD and it is one of the leading causes of death among the elderly in developed countries. Liposomes are biocompatible and biodegradable spherical phospholipid bilayer vesicles that can enclose various compounds. Several functional groups can be attached on the surface of liposomes in order to achieve long-circulating target-specific liposomes. Liposomes can be utilized as drug carriers and vehicles for imaging agents. Positron emission tomography (PET) is a non-invasive imaging method to study biological processes in living organisms. In this study using nucleophilic 18F-labeling synthesis, various synthesis approaches and leaving groups for novel PET imaging tracers have been developed to target AD pathology in the brain. The tracers were the thioflavin derivative [18F]flutemetamol, curcumin derivative [18F]treg-curcumin, and functionalized [18F]nanoliposomes, which all target Aβ in the AD brain. These tracers were evaluated using transgenic AD mouse models. In addition, 18F-labeling synthesis was developed for a tracer targeting the S1P3 receptor. The chosen 18F-fluorination strategy had an effect on the radiochemical yield and specific activity of the tracers. [18F]Treg-curcumin and functionalized [18F]nanoliposomes had low uptake in AD mouse brain, whereas [18F]flutemetamol exhibited the appropriate properties for preclinical Aβ-imaging. All of these tracers can be utilized in studies of the pathology and treatment of AD and related diseases.

High frequency of the CCR5delta32 variant among individuals from an admixed Brazilian population with sickle cell anemia

Homozygous sickle cell disease (SCD) has a wide spectrum of clinical manifestations. In Brazil, the main cause of death of individuals with SCD is recurrent infection. The CCR5delta32 allele, which confers relative resistance to macrophage-tropic HIV virus infection, probably has reached its frequency and world distribution due to other pathogens that target macrophage in European populations. In the present investigation a relatively higher prevalence (5.1%) of the CCR5delta32 allele was identified, by PCR amplification using specific primers, in 79 SCD patients when compared to healthy controls (1.3%) with the same ethnic background (Afro-Brazilians). Based on a hypothesis that considers SCD as a chronic inflammatory condition, and since the CCR5 chemokine receptor is involved in directing a Th1-type immune response, we suggest that a Th1/Th2 balance can influence the morbidity of SCD. If the presence of the null CCR5delta32 allele results in a reduction of the chronic inflammation state present in SCD patients, this could lead to differential survival of SCD individuals who are carriers of the CCR5delta32 allele. This differential survival could be due to the development of less severe infections and consequently reduced or less severe vaso-occlusive crises.

A direct contact between astrocyte and vitreous body is possible in the rabbit eye due to discontinuities in the basement membrane of the retinal inner limiting membrane

Different from most mammalian species, the optic nerve of the rabbit eye is initially formed inside the retina where myelination of the axons of the ganglion cells starts and vascularization occurs. Astrocytes are confined to these regions. The aforementioned nerve fibers known as medullated nerve fibers form two bundles that may be identified with the naked eye. The blood vessels run on the inner surface of these nerve fiber bundles (epivascularization) and, accordingly, the accompanying astrocytes lie mostly facing the vitreous body from which they are separated only by the inner limiting membrane of the retina. The arrangement of the astrocytes around blood vessels leads to the formation of structures known as glial tufts. Fragments (N = 3) or whole pieces (N = 3) of the medullated nerve fiber region of three-month-old male rabbits (Orictolagus cuniculus) were fixed in glutaraldehyde followed by osmium tetroxide, and their thin sections were examined with a transmission electron microscope. Randomly located discontinuities (up to a few micrometers long) of the basement membrane of the inner limiting membrane of the retina were observed in the glial tufts. As a consequence, a direct contact between the astrocyte plasma membrane and vitreous elements was demonstrated, making possible functional interactions such as macromolecular exchanges between this glial cell type and the components of the vitreous body.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS· and NacyS·) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

Dimensions and dynamics in integrin function

Integrins play crucial roles in cell adhesion, migration, and signaling by providing transmembrane links between the extracellular matrix and the cytoskeleton. Integrins cluster in macromolecular complexes to generate cell-matrix adhesions such as focal adhesions. In this mini-review, we compare certain integrin-based biological responses and signaling during cell interactions with standard 2D cell culture versus 3D matrices. Besides responding to the composition of the matrix, cells sense and react to physical properties that include three-dimensionality and rigidity. In routine cell culture, fibroblasts and mesenchymal cells appear to use focal adhesions as anchors. They then use intracellular actomyosin contractility and dynamic, directional integrin movements to stretch cell-surface fibronectin and to generate characteristic long fibrils of fibronectin in "fibrillar adhesions". Some cells in culture proceed to produce dense, three-dimensional matrices similar to in vivo matrix, as opposed to the flat, rigid, two-dimensional surfaces habitually used for cell culture. Cells within such more natural 3D matrices form a distinctive class of adhesion termed "3D-matrix adhesions". These 3D adhesions show distinctive morphology and molecular composition. Their formation is heavily dependent on interactions between integrin alpha5ß1 and fibronectin. Cells adhere much more rapidly to 3D matrices. They also show more rapid morphological changes, migration, and proliferation compared to most 2D matrices or 3D collagen gels. Particularly notable are low levels of tyrosine phosphorylation of focal adhesion kinase and moderate increases in activated mitogen-activated protein kinase. These findings underscore the importance of the dimensionality and dynamics of matrix substrates in cellular responses to the extracellular matrix.

Functional cellulose microspheres for pharmaceutical applications

Dissolving cellulose is the first main step in preparing novel cellulosicmaterials. Since cellulosic fibres cannot be easily dissolved in water-based solvents, fibres were pretreated with ethanol-acid solution prior to the dissolution. Solubility and changes on the surface of the fibres were studied with microscopy and capillary viscometry. After the treatment, the cellulose fibres were soluble in alkaline urea-water solvent. The nature of this viscous solution was studied rheologically. Cellulose microspheres were prepared by extruding the alkaline cellulose solution through the needle into an acidic medium. By altering the temperature and acidity of the mediumit was possible to adjust the specific surface area and pore sizes of themicrospheres. A typical skin-core structure was found in all samples. Microspheres were oxidised in order to introduce anionic carboxylic acid groups (AGs). Anionic microspheres are more hydrophilic; their water-uptake increased 25 times after oxidation and they could swell almost to their original state (88%) after drying and shrinking. Swelling was studied in simulated physiological environments, corresponding to stomach acid and intestines (pH 1.2-7.4). Oxidised microspheres were used as a drug carriers. They demonstrated a highmass uniformity, which would enable their use for personalised dosing among different patients, including children. The drug was solidified in microspheres in amorphous form. This enhanced solubility and could be used for more challenging drugs with poor solubility. The pores of themicrospheres also remained open after the drug was loaded and they were dried. Regardless of the swelling, the drug was released at a constant rate in all environments.

Oxygen Photoreduction in Cyanobacteria

Cyanobacteria are well-known for their role in the global production of O2 via photosynthetic water oxidation. However, with the use of light energy, cyanobacteria can also reduce O2. In my thesis work, I have investigated the impact of O2 photoreduction on protection of the photosynthetic apparatus as well as the N2-fixing machinery. Photosynthetic light reactions produce intermediate radicals and reduced electron carriers, which can easily react with O2 to generate various reactive oxygen species. To avoid prolonged reduction of photosynthetic components, cyanobacteria use “electron valves” that dissipate excess electrons from the photosynthetic electron transfer chain in a harmless way. In Synechocystis sp. PCC 6803, flavodiiron proteins Flv1 and Flv3 comprise a powerful electron sink redirecting electrons from the acceptor side of Photosystem I to O2 and reducing it directly to water. In this work, I demonstrate that upon Ci-depletion Flv1/3 can dissipate up to 60% of the electrons delivered from Photosystem II. O2 photoreduction by Flv1/3 was shown to be vital for cyanobacteria in natural aquatic environments and deletion of Flv1/3 was lethal for both Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 under fluctuating light conditions. The lethal phenotype observed in the absence of Flv1/3 results from oxidative damage to Photosystem I, which appeared to be a primary target of reactive oxygen species produced upon sudden increases in light intensity. Importantly, cyanobacteria also possess other O2 photoreduction pathways which can protect the photosynthetic apparatus. This study demonstrates that respiratory terminal oxidases are also capable of initiating O2 photoreduction in mutant cells lacking the Flv1/3 proteins and grown under fluctuating light. Photoreduction of O2 by Rubisco was also shown in Ci-depleted cells of the mutants lacking Flv1/3, and thus provided the first evidence for active photorespiratory gas-exchange in cyanobacteria. Nevertheless, and despite the existence of other O2 photoreduction pathways, the Flv1/3 route appears to be the most robust and rapid system of photoprotection. Several groups of cyanobacteria are capable of N2 fixation. Filamentous heterocystous N2- fixing species, such as Anabaena sp. PCC 7120, are able to differentiate specialised cells called heterocysts for this purpose. In contrast to vegetative cells which perform oxygenic photosynthesis, heterocysts maintain a microoxic environment for the proper function of the nitrogenase enzyme, which is extremely sensitive to O2. The genome of Anabaena sp. PCC 7120 harbors two copies of genes encoding Flv1 and Flv3 proteins, designated as “A” and “B” forms. In this thesis work, I demonstrate that Flv1A and Flv3A are expressed only in the vegetative cells of filaments, whilst Flv1B and Flv3B are localized exclusively in heterocysts. I further revealed that the Flv3B protein is most responsible for the photoreduction of O2 in heterocysts, and that this reaction plays an important role in protection of the N2-fixing machinery and thus, the provision of filaments with fixed nitrogen. The function of the Flv1B protein remains to be elucidated; however the involvement of this protein in electron transfer reactions is feasible. Evidence provided in this thesis indicates the presence of a great diversity of O2 photoreduction reactions in cyanobacterial cells. These reactions appear to be crucial for the photoprotection of both photosynthesis and N2 fixation processes in an oxygenic environment.

Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ± 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol™ and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

Distribution of small proteoglycans and glycosaminoglycans in humerus-related articular cartilage of chickens

The expression of components present in the cartilaginous extracellular matrix is related to development, gender, and genotype, as well as to the biomechanical properties of each type of cartilage. In the present study, we analyzed small proteoglycans and glycosaminoglycans present in different cartilages of the chicken wing after extraction with guanidine hydrochloride or papain. Quantitative analysis of glycosaminoglycans showed a larger amount in humeral cartilage (around 200 mg/g tissue) than in articular cartilage of the radius and ulna, with 138 and 80 mg/g tissue, respectively. Non-collagenous proteins isolated were predominantly from cartilage in the proximal regions of the humerus and radius. D4 fractions obtained by ultracentrifugation were separated by DEAE-Sephacel and Octyl-Sepharose chromatography and analyzed by SDS-PAGE. Two bands of 57 and 70-90 kDa were observed for all samples treated with ß-mercaptoethanol. Immunoblotting of these proteins was positive for the small proteoglycans fibromodulin and decorin, respectively. Apparently, the 57-kDa protein is present in macromolecular complexes of 160 and 200 kDa. Chondroitin sulfate was detected in all regions. HPLC analysis of the products formed by chondroitinase AC and ABC digestion mainly revealed ß-D-glucuronic acid and N-acetyl ß-D-galactosamine residues. The 4-sulfation/6-sulfation ratio was close to 3, except for the proximal cartilage of the radius (2.5). These results suggest functional differences between the scapula-humerus, humerus-ulna, and humerus-radius joints of the chicken wing. This study contributes to the understanding of the physiology of cartilage and joints of birds under different types of mechanical stress.

Recombinant viruses as vaccines against viral diseases

Vaccine approaches to infectious diseases are widely applied and appreciated. Amongst them, vectors based on recombinant viruses have shown great promise and play an important role in the development of new vaccines. Many viruses have been investigated for their ability to express proteins from foreign pathogens and induce specific immunological responses against these antigens in vivo. Generally, gene-based vaccines can stimulate potent humoral and cellular immune responses and viral vectors might be an effective strategy for both the delivery of antigen-encoding genes and the facilitation and enhancement of antigen presentation. In order to be utilized as a vaccine carrier, the ideal viral vector should be safe and enable efficient presentation of required pathogen-specific antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its production on a large-scale basis. Several viral vaccine vectors have thus emerged to date, all of them having relative advantages and limits depending on the proposed application, and thus far none of them have proven to be ideal vaccine carriers. In this review we describe the potential, as well as some of the foreseeable obstacles associated with viral vaccine vectors and their use in preventive medicine.

APOA1/C3/A4 gene cluster variability and lipid levels in Brazilian children

Genetic studies have suggested that polymorphisms of genes coding for apolipoproteins are significant determinants of serum lipoprotein and lipid levels in adults. However, only a few studies have investigated the association of these polymorphisms in children. Therefore, in the present investigation we studied the distribution of APOA1 -75 G>A, +83 C>T, APOC3 -482 C>T, -455 T>C and 3238 C>G, and APOA4 Q360H and T347S polymorphisms and their influence on plasma lipoprotein levels in children from a Brazilian northeastern admixed population. The seven polymorphic sites were genotyped in 414 children aged 5 to 15 years (mean 8.9 ± 2.9). The genotypes of the seven polymorphic sites were assessed by PCR-RFLP methods. The frequencies of the less common alleles were, in general, intermediate among parental populations, as expected. Strong linkage disequilibrium was detected between polymorphisms at the APOA1, APOC3 and APOA4 loci in this admixed population sample. Overall the genotype effects seen in adults were weaker or absent in children. The APOC3/-455 and APOA4 T347S variants showed significant effects on HDL cholesterol in girls (P = 0.033 and P = 0.016, respectively). Significantly higher plasma total (P = 0.003) and LDL cholesterol (P = 0.004) levels were observed in boys who were carriers of the 3238G allele at the APOC3/3238 C>G site. These results disclosed an overall absence of associations between these polymorphisms and lipids in children. This finding is not unexpected because expression of the effect of these polymorphisms might depend on the interaction with environmental variables both internal and external to the individual.