The 3-D fluorescence microscope has become the method of choice in biological sciences for living cells study. However, the data acquired with conventional3-D fluorescence microscopy are not quantitatively significant because of distortions induced by the optical acquisition process. Reliable measurements need the correction of theses distortions. Knowing the instrument impulse response, also known as the PSF, one can consider the backward process of convolution induced by the microscope, known as "deconvolution". However, when the system response is not invariant in the observation field, the classical algorithms can introduce large errors in the results. In this thesis we propose a new approach, which can be easily adapted to any classical deconvolution algorithm, direct or iterative, for bypassing the non-invariance PSF problem, without any modification to the later. Based on the hypothesis that the minimal error in a restored image using non-invariance assumption is located near the used PSF position, the EMMA (Evolutive Merging Masks Algorithm) blends multiple deconvolutions in the invariance assumption using a specific merging mask set. In order to obtain sufficient number of measured PSF at various depths for a better restoration using EMMA (or any other depth-variant deconvolution algorithm) we propose a 3D PSF interpolation algorithm based on the image moments theory using Zernike polynomials as decomposition base. The known PSF are decomposed into Zernike moments set and each moment's variation is fitted into a polynomial function, the resulting functions are then used to interpolate the needed PSF's Zernike moments set to reconstruct the interpolated PSF.

The human prion protein (PrP) gene (PRNP) codon 129 (M/V) polymorphism is a susceptibility factor for variant Creutzfeldt-Jakob Disease (vCJD) and a major determinant of clinico-pathological phenotype in sporadic CJD. The role of codon 129 in defining susceptibility and strain transmission properties has been investigated in three lines of transgenic mice that express human PrP. The human PRNP gene has directly replaced the murine version, by gene targeting, and variation at codon 129 has given the three genotype lines (HuMM, HuMV, and HuVV). The genetics of these three mouse lines are otherwise identical, and therefore differences in transmission properties can be directly attributable to the codon 129 genotype. vCJD inoculation has shown that all three codon 129 genotype mice are susceptible with a ranking of transmission efficiency of HuMM>HuMV>HuVV. HuMM mice develop the most widespread neuropathology with features similar to human vCJD. Subclinical infection was noted in each mouse line. These data suggest that the vCJD strain is transmissible to humans of each of the three codon 129 genotypes, implying that non-MM cases of human infection with bovine spongiform encephalopathy (BSE) may exist but with long subclinical incubation periods. Inoculation of material from blood transfusion associated vCJD showed no change in transmission properties suggesting that the threat of a future epidemic of human-to-human vCJD infection has not been increased by adaptation of the vCJD strain. However the route of infection, for example via blood transfusion or surgery, may be more efficient that the original oral route of BSE infection. sCJD is classified into six subgroups according to clinico-pathological features, and defined by codon 129 genotype and electrophoretic mobility type (1 or 2) of disease associated PrPSc (MM1, MM2, MV1, MV2, VV1, VV2). Typical cases from each subgroup have shown specific transmission properties suggesting that the subgrouping is defining separate disease strains. The commonest subgroup (MM1) was the most transmissible and the HuVV mouse line the most susceptible host. These data outline the transmission risk from all sCJD types to recipients of each codon 129 genotype should an infection event occur, and show the significant role of recipient codon 129 genotype in defining the clinical or subclinical state and the success or failure of transmission. This is important for determining individual risk following known exposure, and for modelling the potential of iatrogenic infection from sCJD patients.

Calmodulin is a highly conserved calcium ion binding protein expressed in all eukaryotic species. The 149 amino acid residues in the primary structure are organized in seven α helices with the highly flexible central α helix connecting the two non-cooperative domains of calmodulin. Each domain contains two EF-hand motifs to which calcium ions bind in a cooperative manner, hence the binding of four calcium ions saturate one calmodulin molecule. In the cardiovascular area calmodulin is involved in the activation of cardiac muscle contraction, and mutations that arise in the genetic sequence of the protein often have severe consequences. One such consequential mutation that can arise brings about the replacement of the highly conserved aspartic acid with glycine at position 130 in the amino acid sequence. In this research, the thermal and chemical stability within the C domain of the D130G variant of human calmodulin was investigated using a new method only requiring circular dichroism spectroscopic measurements. Affinity studies within the C domain of the D130G variant of human calmodulin were performed using fluorescence spectroscopy, and the ligands chosen for this purpose were trifluoperazine and p- HTMI. All analytical experiments were performed with the C domain of wild type human calmodulin as a reference. From the new method, it was concluded that the C domain of the D130G variant of human calmodulin has a slightly decreased stability in terms of Tm and Cm values compared to the C domain of wild type human calmodulin. The affinity analyses indicated that neither trifluoperazine nor p-HTMI discriminates between the C domain of the D130G variant of human calmodulin and the C domain of wild type human calmodulin in terms of dissociation constants. The pivotal outcome from this research is that the new method is applicable for determination of the stability parameters Tm and Cm of heat stable proteins.

The main goal of this degree work is the application of methods of multiple criterion assessment of options for the choice of new vehicles. I have used three methods with cardinal information in this degree work. It is possible to classify these methods according to a calculation procedure, which is used for finding a compromise option. I have chosen the method of weighted sum based on the principle of the benefit maximilization, the method TOPSIS based on the distance minimalization from the ideal option, and the method ELECTRE III based on the preferential relation. I have used these methods for determination of the compromise option for the purchase of new vehicles. I have compared vehicle running costs and purchase costs with current costs delivered from a building company and then I have determinated an annual saving of costs. At the end of this degree work I have given renewal models which I have determined through the optimal method in the case of the renewal of worn-out equipment. I have analysed the problem from more views, from the view of the multiple criterion assessment of options, then from the costs view and finally from the view of renewal models.

Background. Esophageal cancer is ranked in the top ten of diagnosed tumors worldwide. Even though improvements in survival could be noticed over the last years, prognosis remains poor. ETS translocation variant 1 (ETV1) is a member of a family of transcription factors and is phosphorylated by mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2). Aim of this study was to evaluate the prognostic role of MK2 and ETV1 in esophageal cancer. Methods. Consecutive patients that underwent surgical resection at the department of surgery at the Medical University of Vienna between 1991 and 2012 were included into this study. After microscopic analysis, tissue micro arrays (TMAs) were created and immunohistochemistry was performed with antibodies against MK2 and ETV1. Results. 323 patients were included in this study. Clinical data was achieved from a prospective patient data base. Nuclear overexpression of MK2 was observed in 143 (44.3%) cases for nuclear staining and in 142 (44.0%) cases a cytoplasmic overexpression of MK2 was observed. Nuclear and cytoplasmic ETV1 overexpression was detected in 20 cases (6.2%) and 30 cases (9.3%), respectively. In univariate survival analysis, cMK2 and nETV1 were found to be significantly associated with patients' overall survival. Whereas overexpression of cMK2 was associated with shorter, nETV1 was associated with longer overall survival. In multivariate survival analysis, both cMK2 and nETV1 were found to be independent prognostic factors for the subgroup of EAC as well. Discussion. Expression of MK2 and ETV1 are prognostic factors in patients, with esophageal adenocarcinoma.

Dermicidina (DCD) é um gene mapeado no cromossomo 12, lócus 12q13.1, e codifica uma proteína de 110 aminoácidos, que sofre um processamento proteolítico, gerando peptídeos ativos. O peptídeo C-terminal (DCD-1L) de 48 aminoácidos tem uma carga -2, e exerce função antibacteriana e antifúngica, e o peptídeo C-terminal splice variante, denominado DCD-SV de 59 aminoácidos, tem carga neutra, e suas propriedades ainda não foram estabelecidas. Neste trabalho são apresentados os resultados da expressão, purificação e sequenciamento da DCD nativa produzida em E. coli BL21 transformada com o vetor pAE-DCD. Na segunda parte são descritas as análises físico-químicas e bioquímicas da interação dos peptídeos sintéticos DCD-1L e DCD-SV com vesículas lipídicas gigantes e vesículas unilamelar grandes sintetizadas com palmitoil-oleoil-fosfatidilcolina. As preferenciais estruturais dos peptídeos foram investigadas por espectroscopia de Dicroísmo Circular. Nossos resultados sugerem que a DCD-SV tem alta propensão para adotar uma estrutura helicoidal permitindo sua inserção e oligomerização em membranas biomiméticas, e possível formação de canais de condutância molecular. / Dermicidin (DCD) is mapped a gene on chromosome 12, locus 12q1.13 whose 110 amino acids protein is proteolytically processed to N and C-terminal peptides. The 48-amino acid C-terminal peptide (DCD-1L) has -2 net charges and display antibacterial and antifungal properties and the 59-amino acid splice variant C-terminal peptide (DCD-SV) has neutral net charge; however, its structure and biological function are unknown. Here we show the results of expression, purification and amino acid sequencing of recombinant DCD protein produced in E.coli transformed with pAE-DCD vector. We also describe the results of physical-chemical and biochemical analyses showing the visible differences between the interactions of DCD-1LL and DCD-SV synthetic peptides with giant unilamellar vesicles and large unilamellar vesciles made of palmitoyl-oleoyl phosphatidylcholine, used as biomimetic membranes. The structural preferences of peptides were analyzed by circular dichroism spectroscopy. Our results suggest that DCD-SV peptide has higher propensity to adopt helicoidal structure enabling it to insert into mimetic membranes, undergo oligomerization and formation of conductance channel.

Mais de 95% de luz ultravioleta (UV) que atinge a superfície da Terra corresponde ao comprimento de onda da luz UVA (315-400 nm). A luz UVA induz no DNA danos pela absorção direta e indireta dos fótons, bem como por intermédio de cromóforos. Pacientes com Xeroderma Pigmentosum Variante (XP-V) possuem mutações na DNA polimerase η (pol eta), que promove a síntese translesão dos danos induzidos pela luz solar. Na ausência dessa polimerase há aumento da mutagênese, provável causa de câncer de pele em pacientes XP-V. O objetivo deste trabalho foi caracterizar os mecanismos de indução de danos no genoma e mutagênese por luz UVA, em células derivadas desses pacientes. Os resultados indicam que a exposição à luz UVA resultou em maior sensibilidade de células XP-V comparadas à linhagem controle. Os níveis de fosforilação da histona H2AX (gerando γH2AX - indicador de indução de danos no genoma) e detecção de danos diretos e indiretos no DNA apresentou um aumento significativo em células XP-V irradiadas com luz UVA. Curiosamente, na ausência de pol eta houve uma redução na capacidade de remoção das lesões formadas. Além disso, a irradiação com luz UVA causou forte bloqueio de replicação do DNA e parada do ciclo celular em fase S em células XP-V, desencadeando importantes respostas mediadas por ATR/Chk1. Surpreendentemente, o antioxidante N-acetilcisteína (NAC) resultou em diminuição da sensibilidade celular, dos níveis de γH2AX, da parada de forquilha de replicação e do ciclo celular, reduziu os efeitos citotóxicos da inibição de ATR, melhorou o reparo de lesões no DNA e preveniu a carbonilação de proteínas em células XP-V irradiadas com luz UVA. Investigamos também a indução de mutagênese nas células irradiadas com luz UVA, através de sequenciamento de exoma de clones celulares. Os dados indicaram um aumento significativo da mutagênese em células XP-V irradiadas comparadas a células controle, e pela avaliação dos tipos de mutações encontradas verificamos uma frequência bastante alta de transições C>T, provavelmente como consequência de replicação errônea de dímeros de pirimidina. Entretanto, também identificamos indução significativa de transversões C>A, provavelmente devido a lesões oxidadas no genoma. Curiosamente, ao compararmos células XP-V com células controle detectamos um aumento desse último tipo de mutação na ausência de pol eta, provavelmente devido a lesões endógenas, produzidas pela oxidação do DNA. Além disso, análises in silico mostraram que células XP-V irradiadas com UVA apresentaram assinatura mutacional similar ao observado para tumores de pele. Os dados claramente indicam que células XP-V são sensíveis a irradiação com UVA e que os danos promovidos no DNA, incluindo aqueles causados por estresse oxidativo, desencadeiam respostas celulares e mutagênese nesses pacientes. Assim, além de apontar que UVA pode gerar efeitos deletérios na pele de pacientes XP-V, nossos dados também contribuem para a compreensão de como esses comprimentos de onda podem atuar em células humanas em geral / More than 95% of ultraviolet-light (UV) that reaches the Earth surface corresponds to UVA wavelengths (315-400 nm). UVA-light induces DNA damage through direct and indirect absorption of photons, as well as, intermediated by chromophores and by oxidation mechanisms. Xeroderma Pigmentosum Variant (XP-V) patients are defective in DNA polymerase η (pol eta) that performs translesion synthesis of sunlight induced DNA damage. Absence of pol eta results in increased mutagenesis, which is probably responsible for high frequency of skin cancer in XP-V patients. The goal of this work was to characterize the mechanisms of UVA-induced genome DNA damage and mutagenesis in cells derived from these patients. The results indicate that UVA irradiation increased cell death of XP-V compared to control cell line. The phosphorylation of the histone H2AX (generating γH2AX, an indicator of genotoxic stress) and DNA damage was highly increased in UVA irradiated XP-V cells. Curiously, however, in the absence of pol eta, there was a reduction in the capacity of cells to remove DNA damage from genome. Moreover, UVA irradiation triggered strong DNA synthesis blockage and cell cycle arrest in S phase, resulting in important responses mediated by the ATR/Chk1 pathway in XP-V cells. Interestingly, the antioxidant N-acetyl cysteine (NAC) resulted in decreased cell sensitivity, γH2AX levels, fork stalling and cell cycle arrest, reduced the cytotoxic effect of ATR inhibition, improved DNA repair and prevented the protein carbonylation in XP-V cells irradiated with UVA. The mutagenesis by UVA-light was also investigated by exome DNA sequencing of cellular clones. The data indicated a significative increase of mutagenesis in XP-V irradiated cells compared to control cells, and the identification of mutation types indicated a high increase of C>T transitions, probably as result of error-prone replication of pyrimidine dimers. Nevertheless, the induction of C>A transversions were also detected, probably due to oxidized DNA damage. Curiously, when XP-V and control cells were compared, in the absence of irradiation, these transversions were also detected, maybe due to endogenous oxidation of DNA. In addition, in silico analyses showed that UVA-irradiated XP-V cells had a mutation signature similar to the observed for skin cancer. The data demonstrate XP-V cells are sensitive to UVA-light and DNA damage, including by oxidative stress, trigger cell responses and induce mutagenesis in these patients. Therefore, besides showing that UVA-irradiation may generate deleterious effects in the skin of XP-V patients, the data also contribute to understand how these light wavelengths may damage human cells in general

A relação evolutiva entre mamíferos e Plasmodium compreende infecções com reflexos adaptativos de ambos os lados. A evasão imune pelo parasita e a aquisição imune do hospedeiro contra antígenos importantes são extremos de um longo processo. Genes e antígenos variantes do parasita funcionam nesse processo desempenhando papel evasivo, de citoaderência e manutenção do processo infeccioso. Hospedeiros podem adquirir imunidade efetiva por vacinas contra antígenos variantes e antígenos de caráter reinvasivo. Testamos aqui três desenhos experimentais visando: 1- Definir se antigenos variantes BIR estão associados a citoadesão em infecção murina e se sua localização é em eritrócitos infectados. 2- O papel de um transgene controlado por um promotor de genes variantes no processo adaptativo infeccioso, frente a hospedeiros que diferem em apenas um único gene. 3- Criação de um modelo vacinal aplicável a qualquer antígeno relevante em infecções pelo gênero Plasmodium. Como resultados obtivemos que: 1- Antigenos BIR não parecem estar relacionados na cito adesão em modelo murino. Identificamos um outro grupo de antígenos com domínio LCCL que talvez desempenhe um papel de ligante. 2- promotores bir podem sofrer modulação em uma única infecção que difiere em somente um gene em hospedeiros. Os efeitos desencadeados foram maior parasitemia, anergia e tolerância imune sem afetar a morbidade da infecção de maneira nociva ao hospedeiro. Esse efeito parece ser mediado por subpopulações parasitarias usando exossomos entre parasita e hospedeiro. 3- Produzimos um sistema funcional de produção de proteínas recombinantes fusionadas a GPI que permite integração em lipossomos para usos vacinais. A prova de princípio foi o uso de antígenos PfRH5-GPI recombinantes em teste vacinal que conseguiram gerar anticorpos com alta atividade inibitória em cultivos de P. falciparum. Tomados em conjunto mostramos que o hospedeiro é capaz influenciar a expressão de transgenes controlados por promotores bir e que proteolipossomos contendo antígenos relevantes, como PfRH5, possuem potencial protetor quando vacinados contra malaria. / The relation between mammals and Plasmodium comprise infections with adaptive reflections for both sides. The immune evasion by parasites and immune acquisition by the host against important antigens are end points of a long process. Variant genes and proteins of the parasite can exert a role in this process by enbling immune evasion, cytoadherence resulting in the maintainence of the infective process. We tested three experimental approaches focusing on the following points:1-Show if variant BIR antigens are associated with cytoadherence during murine infections 2- The role of a transgene under the control of a variant gene promoter in adaptation to infection in hosts which express or not the transgene 3- Creation of a vaccine model applicable to any relevant antigen in infections with Plasmodium. As results we showed that: 1-BIR antigenes are likely not related to cito adhesion in the murine model. Cytoadherence in this model is probably related to exported parasite proteins with LCCL domains 2-bir promoters can be modulated during infection in hosts which which differ in one unique gene.The effects observed in this case was an increase in parasitemia, anergy and immune tolerance without affecting the morbidity of infection in the host. These effects are apparently mediated by parasite subpopulations producing exosomes that signal from the parasite to the host.3- We generated a system for recombinant protein production where antigens are fused to GPI and then integrated onto liposomes for vaccine usage. The proof of principle was the use of recombinant PfRH5-GPI as vaccine which elicited antibodies with strong blocking activity in P. falciparum cultures. Together we have shown that the host environment is capable of modulating the activity of variant bir gene promoters and that proteoliposomes loaded with relevant malarial antigens such as PfRH5, are potentially protective when used as malaria vaccine.

Introdução: A papilomatose recorrente respiratória (PRR) é uma doença benigna de enorme morbidade, com manifestações clínicas muito distintas, causada pelo papiloma vírus humano (HPV) dos tipos 6 e 11. Objetivo: Identificar os diferentes tipos de HPV e suas variantes relacionados com a PRR e estabelecer possível correlação clínica entre o tipo / variante viral e a agressividade da doença. Casuística e Métodos: Estudo prospectivo, realizado com 41 pacientes atendidos no Ambulatório de Laringologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, entre os anos de 2008 e 2015, que apresentaram quadro de PRR. Foi realizada genotipagem viral das biopsias laríngeas e os vírus presentes foram relacionados com a evolução clínica de cada paciente e a agressividade da doença, pelo escore de Derkay, número de cirurgias e traqueostomia. Resultados: Dos 41 pacientes avaliados, a maioria era do sexo masculino (68,29%), e maiores de 12 anos (63,41%). Os menores de 12 anos apresentaram número de cirurgias e escore de Derkay significativamente maiores do que os pacientes mais velhos. Foram identificados somente os HPV dos tipos 6 e 11, na frequência de 73,17% e 26,83%, respectivamente. A agressividade da doença não mostrou relação com o tipo ou a variante do vírus, mas sim com a idade de apresentação clínica da doença. Conclusão: Os tipos de HPV e as suas variantes não apresentaram comportamento clínico mais agressivo na PRR no grupo estudado. Em contrapartida, a idade foi determinante para a agressividade da doença. / Introduction: Recurrent respiratory papillomatosis (RRP) is a benign disease of enormous morbidity that presents very distincts clinical manifestations. It is usually caused by the human papillomavirus (HPV), mostly types 6 and 11. Purpose: To identify the different HPV types and variants related to RRP and to establish a possible correlation between HPV type/variant and viral disease aggressiveness. Casuistic and Methods: This prospective study included 41 patients followed at the Outpatient Clinic of Laryngology of the Medical School of Ribeirão Preto, University of São Paulo, between 2008 to 2015, due to RRP. Virus genotyping was performed on laryngeal biopsies by PCR, and this information was associated to each patient\'s data regarding aggressiveness of the disease and clinical evolution, by Derkay\'s score, number of surgeries and tracheostomy. Results: Most patients were male (68.29%) and older than 12 years-old (63.41%). Patients younger than 12 years had a significantly higher number of surgeries and Derkay score than older patients. In all samples, only HPV 6 and HPV 11 were detected, in a prevalence of 73.17% and 26.83%, respectively. The aggressiveness of the disease was not related to the type or variant of the virus, but to the age of patient when the clinical presentation of the disease occurred. Conclusions: HPV types and variant of HPV showed no more aggressive clinical behavior in recurrent respiratory papillomatosis in the studied group. In contrast, age was crucial to the aggressiveness of the disease.

The finite element method (FEM) has been widely applied to various medical imaging applications over the past two decades. The remarkable progress in high-resolution imaging techniques has allowed FEM to draw great research interests in computing trabecular bone (TB) stiffness from three-dimensional volumetric imaging. However, only a few results are available in literature on applying FEM to multi-row detector CT (MDCT) imaging due to the challenges posed by limited spatial resolution. The research presented here develops new methods to preserve TB structure connectivity and to generate high-quality mesh representation for FEM from relatively low resolution images available at MDCT imaging. Specifically, it introduced a space-variant hysteresis algorithm to threshold local trabecular structure that preserves structure connectivity. Also, mesh generation algorithms was applied to represent TB micro-architecture and mesh quality was compared with that generated by traditional methods. TB stiffness was computed using FEM simulation on micro-CT (µ-CT) and MDCT images of twenty two cadaveric specimens of distal tibia. Actual stiffness of those specimens were experimentally determined by mechanical testing and its correlation with computed stiffness was analyzed. The observed values of linear correlation (r2) between actual bone stiffness and computed stiffness from µ-CT and MDCT imaging were 0.95 and 0.88, respectively. Also, reproducibility of the FEM-based computed bone stiffness was determined from repeat MDCT scans of cadaveric specimens and the observed intra-class correlation coefficient was a high value of 0.98. Experimental results demonstrate the feasibility of application of FEM with high sensitivity and reproducibility on MDCT imaging of TB at distal tibia under in vivo condition.