DC - Teses de Doutoramento
Permanent URI for this collection
Browse
Browsing DC - Teses de Doutoramento by Title
Now showing 1 - 10 of 40
Results Per Page
Sort Options
- Analysis of TIM2 deficiency in the mouse retinaPublication . Valença, Andreia Barbosa; Ruberte París, Jesús; Jorge, Maria Luísa MendesCareful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and progress of several retinopathies, such as diabetic retinopathy. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter and can be regarded as a potential deliverer of a considerable large amount of iron to the retina compared to transferrin, the classical ironcarrier protein. Ferritin can bind to scavenger receptor class A member 5 (Scara5) and T-cell immunoglobulin and mucin-domain 2 (TIM2) receptors and is likely endocytosed. In this study, the presence of TIM2, which remained unknown in the retina, was investigated. Although no human ortholog for mouse TIM2 has been identified, human TIM1 and mouse TIM2 have similar functions. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly expressed in Müller cells, unveiling new aspects of retinal iron metabolism regarding the putative role of TIM2 in this tissue. A knockout mouse for this membrane receptor was generated in order to better understand TIM2 functions in the retina. TIM2 deficiency affected retinal iron metabolism. Iron-loaded ferritin accumulation, probably due to increased ferritin uptake mediated by Scara5, and increased iron uptake by transferrin receptor 1 (TfR1)- transferrin binding led to retinal iron overload. Consequently, increased vascular permeability and blood-retinal barrier (BRB) breakdown were observed, inducing edema of the central retina. Paracellular and transcellular transports were impaired with tight junction integrity loss and increased caveolae number. Two mechanisms seem to be involved in this process: association of iron and ferritin overload with vascular endothelial growth factor (VEGF) overexpression and oxidative stress triggered by reactive oxygen species (ROS) overproduction generated by retinal iron overload. Altogether, these results point to TIM2 as a new key player in iron homeostasis in the mouse retina, possibly modulating cellular iron levels, and a potential target for the treatment of diabetic macular edema.
- Bovine genital campylobacteriosis : new insights into the molecular diagnosis and pathogenesisPublication . Silva, Marta Filipa Serra da; Mateus, Luísa Maria Freire Leal; Silva, Maria Elisabete Tomé Sousa; Duarte, Ana Isabel Simões PereiraC. fetus subsp. venerealis is the pathogen responsible for the Bovine Genital Campylobacteriosis (BGC), a cattle’s venereal disease whose diagnosis relies on the accurate identification of Cfv. Despite its relevance for BCG control and management, the applicability of the molecular methods on the disease diagnosis is controversial whereas the mechanisms underlying the pathogenesis of BGC remain unclear. Therefore, the main objectives of this work were to assess the suitability of different real-time PCR assays to be used in bovine preputial samples for Cfv detection and to further elucidate the virulence potential of Cfv through the genomic characterization of Cfv field isolates. The results showed that the currently used molecular targets for Cfv identification, the parA gene and the insertion sequence ISCfe1, originate a high rate of false-positive results (up to 50 %) in clinical samples. This lack of specificity was also evidenced by the identification of a novel bacterial species in the bovine prepuce, Campylobacter portucalensis, with sequences homologous to parA (98 % identity) and ISCfe1 (94 % identity) sequences of Cfv, hindering the molecular diagnosis of BGC. On the other hand, the absence of the parA gene in a high proportion of Cfv strains (77 %), also invalidate the use of this gene as a diagnostic target for Cfv identification. The identification of three novel Sequence Types (ST) in venerealis subspecies, previously characterized by a high genetic stability, questions the use of Multilocus Sequence Typing (MLST) for subspecies identification. This work additionally evidenced differential features between Cfv and its biovar intermedius, namely in whole-genome Single Nucleotide Polymorphisms (SNPs) and genes encoding genus-specific proteins families. Moreover, the results suggest that a type IV secretion system, previously indicated as involved in Cfv virulence, does not play a crucial role in the pathogenesis. In vitro antimicrobial resistance to streptomycin, penicillin, tetracycline and enrofloxacin was not detected, even in strains encoding two multidrug efflux pumps, CmeABC and YkkCD, revealing that its sole presence in the genome is not enough to provide in vitro antimicrobial resistance. Overall, these results have a significant impact on the molecular diagnosis of BGC and revealed new insights into the Cfv genetic diversity and virulence potential.
- Characterization of insect bite hypersensitivity in a population of Lusitano horses : contribution for future implementation of skin prick tests (SPT) in IBH diagnosisPublication . Pessoa, Vera Purificação Carvalho; Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Ferreira, Manuel BrancoInsect Bite Hypersensitivity (IBH) is a recurrent seasonal pruritic dermatitis affecting many horses worldwide. It is mainly a type I hypersensitivity, but type IV may also occur, to allergens present in the saliva of Culicoides spp. It is a multifactorial disease affecting different breeds, with a variable prevalence. Treatment options are very limited, but being an allergic disease IgE-mediated, there is the possibility of developing specific immunotherapy. This study aimed to characterize IBH in Lusitano horses living in Portugal mainland, through a case control study involving 30 IBH-affected animals and 30 healthy controls. Questionnaires were performed for all the horses involved in the study in order to characterize the studied population. In vivo allergen testing was evaluated by performing skin tests, both skin prick tests (SPT) and intradermal tests (IDT), using 14 specific allergens, including 13 different recombinant (r-) proteins from Culicoides nubeculosus (Cul n) or Culicoides obsoletus (Cul o) salivary glands (termed Cul n 1 to Cul n 11, Cul o 1P and Cul o 2P) as well as Cul n whole body extract (WBE). Moreover, a cluster of 12 of these horses, 6 IBH-affected and 6 from the control group, were also tested with allergens produced in insect cells (Cul n 3Bac, Cul n 4Bac) and barley (Cul n 3 Bar, Cul n 4Bar) as well as Cul o 3 and Cul o whole body extract (WBE). Furthermore, in vitro diagnostic tests have been performed, namely, serum IgEs that were measured by ELISA for the allergens Cul n 3, 4, 9, 10 and Cul o 2, 3, as well as Cul o1P and Cul o2P and in vitro sulfidoleukotriene (sLT) release assay which were carried out with Cul n WBE and Cul o WBE. In our study SPTs presented a higher discriminatory diagnostic potential than IDTs, and should be preferred, being a potential breakthrough in the establishment of more suitable allergen avoidance measures in IBH diagnosis. Regarding the in vitro tests, even though serology measurement of specific IgEs showed that IBH-affected horses had higher serum IgE levels for the significant allergens, sLT release assay performed best. It was also possible to study and identify Culicoides spp found in the stud farms, which may be related to IBH in the studied horses. The most frequently found were Obsoletus group species, C. nubeculosus and C. imicola. This study contributed to improve IBH diagnosis, representing a step forward for the future implementation of locally relevant diagnostic allergen panels and eventually for the establishment of patient tailored, component resolved specific immunotherapy
- Contribution to the discriminant power of some of the variables involved in the staging of severe equine asthma syndromePublication . Simões, Joana de Sousa Azevedo; Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Luís, José Paulo Pacheco SalesSevere Equine Asthma Syndrome (EAS) is a highly prevalent, chronic and recurrent respiratory disease which appears to be related to equine domestication. Due to its insidious nature, treatment may sometimes be frustrating and severe economic loses occur. Genetically susceptible individuals develop airway inflammation, hyperresponsiveness and obstruction when exposed to environments with high concentrations of respirable dust particles, which include mould spores, mites, endotoxins, pollen and other antigenic materials. These hazardous respirable dust concentrations are usually found in traditional equine housing systems, while lower concentrations tend to be present in outdoor systems (pasture). The management of severe EAS essentially requires environmental control to ensure improvement of lung function and in some cases medical treatment with corticosteroids and bronchodilators to ameliorate the clinical signs of airway inflammation and bronchospasm. Considering the clinical importance of this syndrome, the dissertation focuses on further contributing to scientific knowledge of severe EAS. As such, we investigated the influence of lung function tests on the diagnosis and staging of the disease and developed a staging method using only portable equipment, which has the potential of being used in equine ambulatory practice. We also investigated the relation between severe EAS and resistance to gastrointestinal parasites, which had not been, to the author’s knowledge, previously reported in the Lusitano breed horses. This association has been reported in other equine breeds with severe EAS or with other multiple hypersensitivities (MHS). Lastly, because allergen avoidance is fundamental for the remission of severe EAS we examined owner compliance to a set of recommended guidelines for environmental management.
- Contribution to the staging and immunological study of equine recurrent airway obstruction (RAO)Publication . Tilley, Paula Alexandra Botelho Garcia de Andrade Pimenta; Luís, José Paulo Pacheco Sales; Ferreira, Manuel BrancoRecurrent airway obstruction (RAO) is one of the most frequent lung diseases in horses and is similar to human asthma. We characterized equine RAO differential diagnosis (DD) in horses with long term cough and established a parallel between the DD in equine RAO and in human asthma. We correlated clinical, endoscopic, thoracic X-ray and bronchoalveolar lavage fluid scores in horses with RAO to establish relevance of each factor for the characterization of RAO stages in order to suggest a staging method. Cardiovascular effects of RAO were assessed. We also evaluated the response to skin prick tests (SPT) and in vitro allergy tests with common aeroallergens in horses with RAO and characterized RAO in Portugal by identifying relevant allergic factors. The importance of a thorough diagnosis is emphasized, including BAL and respiratory endoscopy, and a DD parallel is made with vocal cord dysfunction in man. A score model for the characterization of RAO stages is suggested. The first ECG and EcoCG values for Lusitano/Lusitano-cross horses are published with subtle changes in the RAO group. In this highly selected population immediate aeroallergen hypersensitivity was significant, allergy being a probable aetiopathogenic mechanism in all RAO group horses.
- Contribution to the study of prognostic factors in canine oral squamous cell carcinomaPublication . Mestrinho, Lisa Alexandra Pereira; Niza, Maria Manuela Grave Rodeia Espada; Peleteiro, Maria da Conceição da Cunha e VasconcelosThe main goal of this clinical work was to contribute to the study of oral squamous cell carcinoma (OSCC) in the dog with regards to molecular and differentiation events, and their relationship with the tumor’s biological behavior – impact on the staging, prognosis or treatment. From the assessment of histological differentiation and proliferation index in a 36 OSCC case series, it was possible to identify a significant association between the proliferating cell nuclear antigen (PCNA) and Ki-67, distribution pattern and histological differentiation. PCNA expression was significantly higher than Ki-67 and a higher proliferation index was associated with poorly differentiated OSCC, resembling what is observed in humans. With regards to intra-oral locations, poorly differentiated tumors were significantly associated with the tonsillar location. Proliferation index was not associated with any of the intra-oral locations evaluated, but it was found that metastatic lymph nodes were significantly associated with OSCC with higher poliferation index. OSCC prognosis was assessed in a 13 case series where the age of the animal and tumor size, location, grade and proliferation were evaluated as post surgical prognostic factors. All cases were standardized – CT staged and clinically managed over a period of 2 years, with bone invasion, no lymphatic invasion nor distant metastasis. The results obtained indicated that grade and proliferation index were related with post operatory prognosis. Animals with high-grade tumors had shorter disease free survival times when compared to intermediate grade tumors. Also animals with high-grade tumors and with high PCNA proliferation index showed a significantly worse post-surgical prognosis. The immunohistochemical expression of the cell adhesion molecule E-cadherin and p63 protein were assessed and related with histological and clinical features at time of diagnosis in a 22 case series. Changes in p63 and E-cadherin expression seem to be frequent molecular events in canine OSCC. It was observed that p63 was overexpressed in all cases and E-cadherin was found to be underexpressed in most cases. Furthermore, p63 expression was related to the proliferation and there were different expression patterns related to tumor grade. None of the histological or immunohistochemical parameters were associated with tumor size, bone invasion or lymph node metastasis. More than the contribution to the knowledge of some of the molecular events involved in this tumor, this work explores the clinical applicability of such markers, especially proliferation, in a practical veterinary clinical setting, ilustrated in this work with one case report. This thesis also explores the potential use of the dog as a model in comparative oncology studies.
- Cytokine gene expression and cellular immune response in dogs with leishmaniosis before and under the two first-line treatment protocols : new insights into the animal diseasePublication . Santos, Marcos André Ferreira; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Gomes, Gabriela Maria SantosCanine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic visceral disease of worldwide concern. The drugs used for its treatment improve the animal’s clinical condition, although, in most cases, the parasites are not completely destroyed. The current study aimed to evaluate the immune response of the dog with leishmaniosis before and during treatment with first-line drugs, by analyzing the profile of cytokines and subsets of CD4+ and CD8+ T-cells in peripheral blood, lymph node and bone marrow. Two groups of six dogs diagnosed with CanL were treated with either miltefosine or meglumine antimoniate in combination with allopurinol. Simultaneously, another group of ten clinically healthy dogs was used as a control group. Upon diagnosis and during the following three months of treatment, clinical signs, hematological and biochemical parameters, urinalysis results and anti-Leishmania antibody titers using IFAT were recorded. Furthermore, peripheral blood, popliteal lymph node and bone marrow mononuclear cells were collected to evaluate the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, these cells were also immunophenotypically analyzed be flow cytometry, using surface monoclonal antibodies anti-CD45, CD3, CD4, CD8, CD25 and intracellular monoclonal antibody anti-nuclear factor FoxP3. Both treatment protocols promoted the remission of clinical signs, normalization of hematological and biochemical parameters and urinalysis values. Sick dogs showed a generalized increase in IFN-γ gene expression and a decrease of IL-2, IL-4, and TGF-β. The expression of IL-12, TNF-α, IL-5, and IL-10 showed variations between groups of dogs and the tissue analyzed. CanL also resulted in an overall increase in the percentage of CD8+ T-cells in all tissues. In the peripheral blood there was also a decrease in CD4+ T-cells and an increase of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T-cells, with the latter also increasing on the bone marrow. CD4+CD25-FoxP3- T-cells showed a marked decrease in blood and bone marrow. During treatment, a trend towards normalization of cytokine gene expression and T-cell subsets was observed. However, high levels of IFN-γ gene expression were still observed in all tissues. In turn, the treatments caused an increase in the percentage of CD4+CD25+FoxP3+ and a decrease in CD8+CD25-FoxP3- T-cells, leading to normalization of CD4+ and CD8+ T cells in all tissues. Furthermore, the effect of treatment on gene expression of cytokines that were not significantly altered by infection indicates that these combined treatment protocols directly affect cytokine production. Both combined treatments are effective in remitting clinical sings and appear to influence the dog’s immune response, sustaining a pro-inflammatory immune environment while promoting the normalization of T-cell subsets. These findings indicate that L. infantum may be able to manipulate elements of the dog's immune system to avoid differentiating an efficient protective response, preventing the rapid development of severe pathology while ensuring the parasite’s survival and securing the possibility of several transmission cycles. Allied to these results, other studies carried out in collaboration with the working group on the role of neutrophils, hepatocytes and Kupffer cells in CanL, as well as the evaluation of treatment in feline leishmaniosis, have allowed to enhance the knowledge in the area of animal leishmaniosis.
- Cytological endometritis in dairy cattle : new insights into pathogenesis, diagnosis and treatmentPublication . Pereira, Gonçalo da Silva; Silva, Maria Elisabete Tomé Sousa; Costa, Luís Filipe Lopes daABSTRACT - In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, with profound detrimental effects on subsequent fertility. Despite the known role of adipokines regulating metabolism and inflammation, the association of adipokine signaling with SCE is still poorly understood. Moreover, the effect of SCE on endometrial transcription was mainly determined from biopsy-derived whole tissue, thus masking effects on cell type-specific gene transcription. In addition, despite the recognized improvement in immune function following n-3PUFA supplementation, the therapeutic potential of these fatty acids for SCE control is still to be determined. Therefore, the main objectives of this work were to assess the relationship between adipokines and SCE (Chapter III), to elucidate the effects of progesterone and SCE in the transcription profiles of endometrial glandular, luminal and stromal cells (Chapters IV, V), and the effects of feeding rumen-protected n-3PUFA on endometrial homeostasis and fertility in postpartum dairy cows (Chapter VI). The results showed that adiponectin in plasma and uterine fluid in addition to chemerin uterine fluid have high discriminatory power for the diagnosis of SCE. This work additionally evidenced that progesterone and SCE affect endometrial transcriptomic profiles in a cell type-specific manner. This work also evidenced that the previous presence of immune cells is still impacting the transcriptome of endometrial cells at the end of the voluntary waiting period and that recovery or persistence of inflammation is associated with transcription patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity. Furthermore, despite having no apparent effect on the endometrial inflammatory status at the end of the voluntary waiting period, dietary supplementation with n-3PUFA decreased calving to conception interval. Overall, this thesis establishes adiponectin as a suitable biomarker of SCE, able to predict the risk of persistence of inflammation in postpartum dairy cows, provides new insights into the persistence and recovery of SCE, thus presenting putative alternative therapeutic strategies. Moreover, this work substantiates dietary n-3PUFA as valid nutraceuticals to improve reproductive parameters in postpartum dairy cows.
- Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discoveryPublication . Sequeira, Ana Filipa Pereira; Fontes, Carlos Mendes Godinho de Andrade; Vincentelli, Renaud; Guerreiro, Catarina Isabel Proença DuarteAnimal venoms are complex mixtures of biologically active molecules that, while presenting low immunogenicity, target with high selectivity and efficacy a variety of membrane receptors. It is believed that animal venoms comprise a natural library of more than 40 million different natural compounds that have been continuously fine-tuned during the evolutionary process to disturb cellular function. Within animal venoms, reticulated peptides are the most attractive class of molecules for drug discovery. However, the use of animal venoms to develop novel pharmacological compounds is still hampered by difficulties in obtaining these low molecular mass cysteine-rich polypeptides in sufficient amounts. Here, a high-throughput gene synthesis platform was developed to produce synthetic genes encoding venom peptides. The final goal of this project is the production of large libraries of recombinant venom peptides that can be screened for drug discovery. A robust and efficient Polymerase Chain Reaction (PCR) methodology was refined to assemble overlapping oligonucleotides into small artificial genes (< 500 bp) with high-fidelity. In addition, two bioinformatics tools were constructed to design multiple optimized genes (ATGenium) and overlapping oligonucleotides (NZYOligo designer), in order to allow automation of the high-throughput gene synthesis platform. The platform can assemble 96 synthetic genes encoding venom peptides simultaneously, with an error rate of 1.1 mutations per kb. To decrease the error rate associated with artificial gene synthesis, an error removal step using phage T7 endonuclease I was designed and integrated into the gene synthesis methodology. T7 endonuclease I was shown to be highly effective to specifically recognize and cleave DNA mismatches allowing a dramatically reduction of error frequency in large synthetic genes, from 3.45 to 0.43 errors per kb. Combining the knowledge acquired in the initial stages of the work, a comprehensive study was performed to investigate the influence of gene design, presence of fusion tags, cellular localization of expression, and usage of Tobacco Etch Virus (TEV) protease for tag removal, on the recombinant expression of disulfide-rich venom peptides in Escherichia coli. Codon usage dramatically affected the levels of recombinant expression in E. coli. In addition, a significant pressure in the usage of the two cysteine codons suggests that both need to be present at equivalent levels in genes designed de novo to ensure high levels of expression. This study also revealed that DsbC was the best fusion tag for recombinant expression of disulfide-rich peptides, in particular when expression of the fusion peptide was directed to the bacterial periplasm. TEV protease was highly effective for efficient tag removal and its recognition sites can tolerate all residues at its C-terminal, with exception of proline, confirming that no extra residues need to be incorporated at the N-terminus of recombinant venom peptides. This study revealed that E. coli is a convenient heterologous host for the expression of soluble and potentially functional venom peptides. Thus, this novel high-throughput gene synthesis platform was used to produce ~5,000 synthetic genes with a low error rate. This genetic library supported the production of the largest library of recombinant venom peptides constructed until now. The library contains 2736 animal venom peptides and it is presently being screened for the discovery of novel drug leads related to different diseases.
- Discovering novel carbohydrate-active enzymes in the cellulosome of anaerobic bacteriaPublication . Fernandes, Vânia Ondina PedroCarbohydrate-active enzymes (CAZymes) include a range of enzymes that, in nature, make, break or modify glycosidic bonds. CAZymes act on highly recalcitrant polysaccharides, such as cellulose and hemicellulose, and often exhibit a modular architecture including catalytic domains fused through flexible linker regions to non-catalytic domains such as carbohydrate-binding modules (CBMs). In some anaerobic bacteria these enzymes can associate in high molecular mass multi-enzyme complexes termed cellulosomes. Cellulosomal organisms express a vast repertoire of plant cell wall degrading enzymes and constitute a promising source for the discovery of novel CAZymes. Presently, an exponential accumulation of genomic and metagenomic information is observed while the identification of the biological role of both genes and proteins of unknown function is sorely lacking. In addition, for most of the known CAZymes, structure and/or biochemical characterization is missing. In this study we have developed innovative approaches for the discovery of novel CAZymes in cellulosomal bacteria and provide a detailed biochemical characterization of some of those enzymes. A high-throughput platform was designed for cloning, expression and production of recombinant cellulosomal proteins in Escherichia coli, aiming at looking for novel cellulosomal CAZymes encoded in the genomes of Clostridium thermocellum and Ruminococcus flavefaciens. As a result, a series of novel prokaryotic expression vectors (pHTP) were constructed to allow ligation-independent cloning with high levels of soluble recombinant protein production. In addition, to allow total automation of the procedure, both novel cell culture media and protein purification methods have been established. The platform allowed the production of 184 cellulosomal proteins of unknown function that after the implementation of an enzyme discovery screen lead to the discovery of a novel family of α-Larabinofuranosidases. In order to achieve recombinant soluble expression in E. coli, novel fusion tags were designed and incorporated into pHTP-derivatives. Both Rf1 and Rf47 tags, derived from cellulosomal components, were shown to display a high capacity to enhance protein solubility, as fusion proteins containing both these tags were expressed at high levels and in the soluble form in E. coli. CBMs were confirmed to affect the catalytic activity of appended CAZymes, as it was illustrated by the CBM32 of CtMan5A. This work revealed that members of family 35 CBM have the capacity to bind β-mannose-containing polymers. The biochemical characterization of PL1A, PL1B and PL9 reported here describes the pectinolytic activity expressed by C. thermocellum cellulosome. These enzymes are appended to CBMs that display considerable ligand promiscuity. The application of β- glucanases in animal feed supplementation was tested either in the free state or while associated in mini-cellulosomes. This study revealed that β-1,3-1,4-glucanases and not β-1,4-glucanases are necessary to improve the nutritive value of barley-based diets for broilers. In addition, it was shown that mini-cellulosomes designed to improve the efficacy of exogenous enzymes used for feed supplementation require an effective mechanism to protect linker regions from proteolytic cleavage.