Dissertations / Theses on the topic 'SgA protein'

Orientador: Tomomasa Yano
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Mestrado
Microbiologia
Mestra em Genética e Biologia Molecular

Understanding the functional impact of physical interactions between proteins and DNA on gene expression is important for developing approaches to correct disease-associated gene dysregulation. I conducted a systematic, functional genetic analysis of protein-DNA interactions in the promoter region of the yeast ribonucleotide reductase subunit gene RNR3. I measured the transcriptional impact of systematically perturbing the major transcriptional regulator, Crt1, and three X-box sites on the DNA known to physically bind Crt1. This analysis revealed interactions between two of the three X-boxes in the presence of Crt1, and unexpectedly, a significant functional role of the X-boxes in the absence of Crt1. Further analysis revealed Crt1- independent regulators of RNR3 that were impacted by X-box perturbation. Taken together, these results support the notion that higher-order X-box-mediated interactions are important for RNR3 transcription, and that the X-boxes have unexpected roles in the regulation of RNR3 transcription that extend beyond their interaction with Crt1.

The focus of this thesis is the early detection of stress in the environment. It has been proposed that studies on the cellular level would detect stress reactions earlier in time compared to common physiological methods. In a series of experiments we investigated how different stress factors, both natural and introduced by man, affect levels of stress proteins. One- and two-dimensional gels were used to determine individual proteins and families of proteins. The two-dimensional gels were also used in a proteomic approach, were the presence and absence of proteins after treatment was observed, and the protein expression signatures (PES) were identified.

Baltic Mytilus edulis was used in all experiments and it is evident that earlier observed differences in physiological rates and pollution sensitivity, compared to marine mussels, is also manifested as lower concentrations of stress proteins after exposure to copper and cadmium. When the Baltic mussels were allowed to acclimate for one month the difference decreased, suggesting an environmentally induced difference (paper I). Pre-exposure to heat before exposure to either a second heat-shock or cadmium was found to enhance the levels of HSP70 and thus tolerance, significantly (paper II). Exposure to a mixture of stress factors (PCB, copper and lowered salinity) revealed synergistic, additive and antagonistic effects in induction of 6 different stress proteins. When analyzing a large number of proteins it was shown that it is possible to identify PES with this technique, and we hypothesize that it could be possible to separate responses to mixtures of stress factors (Papers III and IV). Different techniques were also applied to analyze the protein expression pattern when mussels were exposed to PAH- and PCB-fractions extracted from Baltic Sea sediments. In this experiment the protein assays were accompanied by physiological measurements. All methods indicated stressed conditions, but the variation between individual mussels within treatments was smaller in terms of protein response than for physiological parameters (paper V). It is concluded that measuring the induction of stress proteins is a reliable way to detect stressful conditions. Proteins visualized on a one dimensional gel give a “gross” picture of an organism’s condition. The major challenge is to identify the origin and severity of the elucidated stress response. Further mapping of two-dimensional gels suggested that protein patterns are specific to type and level of stress.

A most important future step is to establish links between sub-cellular protein response to well known physiological effects. This should include long term experiments where altered protein expression signatures are linked to life history characteristics like survival, growth and reproductive success.

Serum amyloid A protein (SAA) is an acute phase protein that has recently gained increasing interest as a potential marker for disease and treatment monitoring. We investigated SAA and CRP levels in (a) patients with various common infectious diseases (n=98), (b) patients with pyelonephritis (n=37) versus patients with cystitis (n=32), (c) healthy individuals of varying ages (n=231), (d) very immature newborn infants with or without nosocomial infections (NIs) (n=72) and (e) patients with bacterial infections treated with cefuroxime (n=81).

SAA significantly correlated with CRP in viral as well as in bacterial infections (for the total group: r²=0.757, p<0.0001) and showed a systemic inflammatory response in 90% of the patients with cystitis as compared with 23% for CRP. Equally high efficiencies (0.96 and 0.94 for SAA and CRP, respectively) were observed in discriminating between pyelonephritis and cystitis. SAA and high sensitive (hs) CRP were lower in umbilical cords (p<0.0001) and higher in elderly adults (p<0.0001-0.03) than in the other age groups; higher in immature newborn infants than in term infants; and higher in the NI group than in the non-NI group. Interindividual variabilities of the time course of the biomarkers SAA and CRP were considerable. Because of the smoothed distribution of SAA and CRP (i.e. elevations were both essentially unchanged during the first 3 days of cefuroxime treatment), these markers were not useful when deciding parenteral-oral switch of therapy, which occurred within this time period in most cases.

SAA is a sensitive systemic marker in cystitis. SAA and hsCRP in umbilical cord blood are close to the detection limit and increase with age. They increase in relation to NI in very immature newborn infants and might therefore be used in diagnosis and monitoring. Finally, SAA and CRP in adults with bacterial infections could not predict an early parenteral-oral switch of antimicrobial therapy.

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

p53, known as the “guardian of the genome”, is the most well-characterized tumor suppressor gene. The central role of p53 is to prevent genome instability. p53 is the central node in an incredibly elaborate genome defense network for receiving various input stress signals and controlling diverse cellular responses. The final output of this network is determined not only by the p53 protein itself, but also by other p53 cooperating proteins. p300 and CBP (CREB-Binding Protein) act as multifunctional regulators of p53 via acetylase and ubiquitin ligase activities. Prior work in vitro has shown that the N-terminal 595 aa of p300 encode both generic ubiquitin ligase (E3) and p53-directed E4 functions. Analysis of p300 or CBP-deficient cells revealed that both coactivators were required for endogenous p53 polyubiquitination and the normally rapid turnover of p53 in unstressed cells. Unexpectedly, p300/CBP ubiquitin ligase activities were absent in nuclear extracts and exclusively cytoplasmic. In the nucleus, CBP and p300 exhibited differential regulation of p53 gene target expression, C-terminal acetylation, and biologic response after DNA damage. p300 activated, and CBP repressed, PUMA expression, correlating with activating acetylation of p53 C-terminal lysines by p300, and a repressive acetylation of p53 lysine-320 induced by CBP. Consistent with their gene expression effects, CBP deficiency augmented, and p300 deficiency blocked, apoptosis after doxorubicin treatment. Subcellular compartmentalization of p300/CBP’s ubiquitination and transcription activities reconciles seemingly opposed functions—cytoplasmic p300/CBP E4 activities ubiquitinate and destabilize p53, while nuclear p300/CBP direct p53 acetylation, target gene activation, and biological outcome after genotoxic stress. p53 is a prominent tumor suppressor gene and it is mutated in more than 50% of human tumors. Reactivation of endogenous p53 is one therapeutic avenue to stop cancer cell growth. In this thesis, we have identified S5as a critical regulator of p53 degradation and activity. S5a is a non-ATPase subunit in the 19S regulatory particle of the 26S proteasome. Our preliminary data indicates that S5a is required for p53 instability and is a negative regulator of p53 tranactivation. As a negative regulator of p53, S5a may therefore also represent a new target for cancer drug development against tumors that specifically maintain wild type p53.

Protein quality control mechanisms are vital in maintaining correct levels of functional proteins in a crowded intracellular milieu. These mechanisms recognise proteins in their non-native state and triage such candidates for either rescue, or channel them towards pathways that lead to their degradation. In the case of secretory and membrane proteins that mislocalize to the cytosol (MLPs), and of newly synthesised tail-anchored (TA) membrane proteins, their exposure of otherwise buried hydrophobic residues necessitates a mechanism of shielding such residues from the aqueous cytoplasm. This task is carried out by the collective actions of SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the heterotrimeric BAG6 (BCL2-associated athanogene 6) complex, that contribute to a SGTA/BAG6 quality control cycle that can direct hydrophobic substrates towards either ubiquitination and proteasomal degradation or productive membrane insertion. SGTA is a modular protein consisting of three domains, an N-terminal dimerisation domain, a central TPR domain, and a substrate binding C-terminal domain. The aim of this thesis is to characterise full-length SGTA using a range of biophysical techniques, with an emphasis to understand its C-terminal domain, molecular details of which remain elusive. In addition, this work aims to uncover how SGTA interacts with its hydrophobic substrates, underlying its role in enforcing cytosolic quality control. To this end, a combination of nuclear magnetic resonance (NMR) spectroscopy and native mass spectrometry experiments have identified a constrained conformation of SGTA in solution mediated by C-terminal dimerisation, and circular dichroism (CD) spectroscopy has revealed the presence of alpha helical regions within the C-terminal domain. Furthermore, fluorine-19 NMR has been used to investigate the interaction of TA proteins with the C-terminal domain of SGTA. Finally, results presented herein establish that SGTA interacts with the intrinsic proteasomal ubiquitin receptor Rpn13 via a two-carboxylate clamp mode of molecular recognition, and this interaction has been characterised by solution NMR spectroscopy, size exclusion chromatography, isothermal titration calorimetry (ITC) and mutagenesis experiments, to uncover details of a potential SGTA/BAG6 quality control cycle operating at the 19S regulatory particle of the proteasome.

In the urchin Strongylocentrotus purpuratus, the expression of the Spec3 gene is associated with the growth of cilia, one of the first morphogenetic events during development. The product of this gene was characterized using an antiserum raised against a peptide corresponding to the predicted amino-terminal portion of the protein. This thesis describes the localization of the Spec3 protein at different stages during embryonic development. Immunocytochemistry indicated that the protein is associated with cilia and Golgi complexes of ectodermal cells. Agents that inhibit protein synthesis and Golgi secretion also altered its normal distribution. Fractionation of cilia and immunoblotting indicate that the protein is associated with the ciliary axoneme and that it behaves as a large aggregate.

In this thesis I describe the construction of a cDNA library of maternal mRNA from the sea-urchin Arbacia punctulata and the subsequent isolation of a cDNA clone for the cylin protein using the technique of hybrid-arrest of translation. The cyclin protein was originally identified in sea-urchin embryos as a protein that was strongly synthesised after fertilisation but destroyed at each cell division (Evans et al, 1983). The DNA sequence of the putative cyclin clone was determined. It contains an open reading frame for a protein of M_r46,000 which bears significant homology to the central region of the sequence of two other cyclin mRNAs found in eggs of the clam Spissula solidissima. The cyclin clone was subcloned into a T7 RNA polymerase transcription vector and the in vitro translation product of the encoded mRNA found to be a protein of apparent M_r56,000 on a 1-D SDS acrylamide gel, which co-migrated with the in vivo synthesised cyclin protein. When the in vitro transcript was micro-injected into Xenopus Aevis oocytes it caused germinal vesicle breakdown, indicative of entry into meiosis, suggesting a role for cyclin in the control of the cell-cycle.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

Neste trabalho estudou-se a encapsulação de proteínas com diferentes pesos moleculares na sílica mesoporosa ordenada SBA-15 para avaliar sua aplicação como adjuvante imunológico. Para tanto, as proteínas IgG (150 kDa) e BSA (66,5 kDa) foram incorporadas à sílica mesoporosa com poros expandidos. Primeiramente estudou-se a dilatação dos poros utilizando-se um agente dilatador de estrutura no processo de síntese, através da preparação de amostras com diferentes quantidades de triisopropilbenzeno (TIPB). Resultados de isoterma de adsorção de nitrogênio (NAI) e espalhamento de raios X a baixo ângulo (SAXS) revelaram um aumento no diâmetro médio de poros da ordem de 23% e uma rede de poros mais desordenada. Para se ter uma estimativa das dimensões das proteínas, medidas de SAXS foram feitas e indicaram que ambas têm dimensões que permitiriam sua incorporação nos poros da SBA-15. As amostras com poros dilatados foram então utilizadas para a incorporação das proteínas IgG e BSA em solução tampão fosfato salina (PBS). Os resultados indicaram o preenchimento dos microporos pela solução de PBS com valor superior a 95%. Quanto ao preenchimento de mesoporos, observou-se maior variação no volume de poros e área superficial, para o material com maior diâmetro médio de poros, atingindo valores em torno de 80 % e 90%, respectivamente. Não houve diferença significativa nestas porcentagens entre a incorporação das duas proteínas, indicando que as proteínas devem estar encapsuladas na macroporosidade da sílica, obstruindo a entrada dos mesoporos. Um modelo teórico foi utilizado para analisar as medidas de SAXS e ele confirmou o preenchimento dos microporos e a presença da proteína nos mesoporos da sílica. Utilizando a técnica analítica de Particle Induced X-ray Emission (PIXE), o conteúdo de silício em fezes de camundongos isogênicos foi avaliado em função do tempo, com o intuito de analisar a liberação de sílica pelo organismo dos camundongos. Observou-se que após a primeira administração de sílica os camundongos inoculados por via oral liberaram uma quantidade de silício similar à liberada pelos camundongos controle, que receberam apenas a solução de PBS. Após a segunda administração os camundongos inoculados com sílica por via oral liberaram uma quantidade de silício superior à dos camundongos controle, devido à morte dos macrófagos e outras células que atuam na resposta imunológica, evidenciando a eliminação de silício pelo organismo. Um estudo paralelo de controle de qualidade foi realizado em amostras de SBA-15 preparadas pela Cristália Produtos Químicos Farmacêuticos Ltda. a fim de garantir a reprodutibilidade deste material, que será comercialmente utilizado para a formulação de vacina oral contra Hepatite B, produzida pelo Instituto Butantan. Os resultados mostraram propriedades texturais reprodutíveis das amostras produzidas pela Cristália indústria farmacêutica.
In this work we studied the encapsulation of proteins with different molecular weights into SBA-15 ordered mesoporous silica to evaluate its application as immunological adjuvant. Hence, the IgG protein (150 kDa) and BSA (66.5 kDa) were incorporated into the mesoporous silica with expanded pores. First we studied the expansion of the pores using a structure swelling agent in the synthesis process, by preparing samples with different amounts of triisopropylbenzene (TiPB). Results of Nitrogen Adsorption Isotherm (NAI) and Small Angle X-Ray Scattering (SAXS) showed an increase in average pore diameter of the order of 23% and a more disordered pore network. To provide an estimate of the size of proteins, SAXS measurements were performed and indicated that both have dimensions that allow its incorporation in the pores of SBA-15. These samples were used for the incorporation of IgG and BSA proteins in phosphate buffered saline (PBS) solution. The results indicated a filling of the micropores by PBS solution larger than 95%. Concerning the filling of mesopores, we observed a larger variation in pore volume and surface area for the material with highest average pore diameter, reaching values around 80% and 90%, respectively. There was no significant difference in these percentages between the incorporation of the two proteins, indicating that the proteins could be encapsulated in the silica macroporosity, blocking the entry of mesopores. A theoretical model was used to analyze the SAXS measurements and it confirmed the filling of micropores and the presence of protein in the silica mesopores. Using the analytical technique \"Particle-Induced X-ray Emission\"(PIXE), the silicon content in mice feces was evaluated as a function of time, in order to analyze the release of silica from the organism of the mice. We observed that after the first silica administration the isogenic mice inoculated by oral administration released a similar amount of silicon compared with the amount released by the isogenic control mice, which received only PBS solution. After the second oral administration, the mice inoculated with silica released a higher amount of silicon than the control mice, due to the death of macrophages and other cells that act on the immune response, indicating the elimination of silicon by the organism. A parallel study about quality control was performed on SBA-15 samples prepared by Cristália Produtos Químicos Farmacêuticos Ltda., to ensure the reproducibility of this material which will be used commercially for the formulation of an oral vaccine against hepatitis B, produced by the Butantan Institute. The results showed reproducible textural properties of the samples produced by the Cristália pharmaceutical industry.

@font-face { font-family: "Arial"; }@font-face { font-family: "ＭＳ 明朝"; }@font-face { font-family: "Calibri"; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0in 0in 0.0001pt; text-indent: 0.5in; line-height: 200%; font-size: 11pt; font-family: "Times New Roman"; }p.MsoBodyText, li.MsoBodyText, div.MsoBodyText { margin: 0in 0in 6pt; text-indent: 0.5in; line-height: 200%; font-size: 11pt; font-family: "Times New Roman"; }span.BodyTextChar { font-family: Calibri; }div.Section1 { page: Section1; } A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica has broad antimicrobial activity in tryptone peptone rich medium. This protein, which we call ‘escapin’, belongs to an L-amino acid oxidase family. The goals of my project were 1) to determine the products of escapin’s oxidation of its main substrate L-lysine, 2) to characterize the antimicrobial effects of escapin’s products, and 3) determine bactericidal mechanisms of action of these products. Escapin is a powerful bactericidal agent against several bacteria species including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio harveyi. Escapin operates through a two-step process: 1) deamination of L-amino acids (especially L-lysine) by enzymatic activity to produce escapin intermediate products of L-lysine (EIP-K), hydrogen peroxide, and ammonia; and 2) EIP-K simultaneously reacts with hydrogen peroxide to generate escapin end products (EEP-K). EIP exists as an equilibrium mixture of the linear a-keto analogue of lysine and its cyclic forms, and the relative amount of the linear form increases with pH decreases. The powerful bactericidal effect of escapin requires the simultaneous presence of hydrogen peroxide and EIP-K in weak acidic conditions, which suggests that linear form of EIP-K with hydrogen peroxide is responsible for the bactericidal effect of escapin. Using E. coli MC4100 as a model, the mechanism of action of escapin was examined. Brief treatment with EIP-K + H2O2, but not EIP-K or H2O2 alone, causes irreversible DNA condensation with a time course similar to the bactericidal effect. A mutant strain resistant to EIP-K + H2O2 was isolated, and a single point mutation was found in the oxidative stress regulator gene (oxyR). Through a complementary assay, it was shown that wild type E. coli is conferred resistance to EIP-K + H2O2 by carrying mutated oxyR plasmid. Furthermore, in this bactericidal effect, heat or cold shock does not substitute for hydrogen peroxide induced oxidative stress. Thus, escapin’s powerful bactericidal effect may be through irreversible DNA condensation mediated through hydrogen peroxide generating an oxidative stress response, but the pathway mediating EIP-K’s synergistic effect is still unclear.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
The movement protein MP from bipartite geminivirus (begomovirus) facilitates the cell-to-cell and long-distance transport of viral DNA in addition to affecting viral pathogenicity. To identify host factors that interact with MP, initially a cDNA library prepared from CaLCuV (Cabbage leaf curl virus)-infected Arabidopsis leaf mRNA was generated in a pEXPAD502 vector. To select for interactions between the bait BD-MP and cDNA library-encoded proteins, double transformants (BD-MP+ cDNA-AD) were plated on medium lacking histidine but supplemented with 3-aminotriazole (3-AT). From 5 x 105 transformants screened, two clones, encoding AtEXL3 (Exordium Like 3), a cell wall protein, and AtRPS5A, ribosomal protein S5A, displayed histidine/adenine auxotrophy and activate LacZ expression, on X-gal indicator plates. Expression of a full-length RPS5A cDNA fused to the Gal4 activation domain in yeast carrying BD-MP promoted growth of the double transformants in the absence of histidine and presence of 3-AT, in addition to activating high levels of LacZ expression. Furthermore, the interaction between MP and RPS5A was confirmed in vitro by pull down assays. Analysis of RPS5A gene expression by qRT-PCR demonstrated that the accumulation of rpS5A transcripts is suppressed by geminivirus infection. Based on these results and others, a functional model for the MP-RPS5A interaction is discussed.
A proteína de movimento (MP) de geminivírus bissegmentados (begomovirus) facilita o movimento célula-célula, bem como o movimento a longas distâncias do DNA viral, além de influenciar na patogenicidade viral. Com o objetivo de identificar fatores do hospedeiro que interagem com MP, inicialmente foi construída uma biblioteca de cDNA a partir de mRNAs de folhas de Arabidopsis infectadas com CaLCuV (Cabbage leaf curl virus) no vetor pEXPAD502. Para selecionar por interações entre a proteína quimérica BD-MP e proteínas codificadas pelos cDNAs da biblioteca, transformantes duplos (BDMP+ cDNA-AD) foram plaqueados em meio deficiente de histidina e suplementados com 3-amino triazol (3-AT). De um total de 5 x 105 transformantes escrutinados, dois clones, codificando EXL3 (Exordium Like 3), uma proteína de parede celular, e RPS5A, proteína ribossomal S5A, apresentaram auxotrofia a histidina e expressão do gene repórter LacZ, em placas indicadoras de X-gal. Expressão do cDNA completo de RPS5A fusionado ao domínio de ativação de Gal-4 em leveduras, carreando BD-MP, promoveu crescimento dos transformantes na ausência de histidina e presença de 3-AT, além de ativar altos níveis de expressão de LacZ.. Além disso, a interação entre MP e RPS5A foi confirmada in vitro por ensaios de pull down. Análises de expressão do gene RPS5A por meio de qRT-PCR demonstraram que o acúmulo de seus transcritos é reprimido por geminivírus. Baseado nestes resultados e de outros, um modelo funcional para interação de MP com RPS5A é discutido.

In this thesis empirical relationships and modeling tools are used to describe the relationship between human activities and meso- and large scale riverine N fluxes from land to sea. On a large scale Paper I showed that by only having knowledge about human population size and runoff one can estimate the riverine export of DIN (r2= 0.76). In Paper II we included two other important anthropogenic N inputs, i.e. atmospheric deposition and primary emission (PE) from animals. In most of the catchments the PE from animals were larger than the PE from humans. Hence, development of livestock is important and increased animal protein consumption by humans might increase the riverine N export. Scenario analysis (Paper II) show that climate change is expected to both decrease and increase the riverine N export depending on which part of the catchment is modeled. In the southern and eastern parts of the Baltic Sea catchment there is large potential for N reductions from point sources (Papers III & V). The diffuse sources are more difficult to decrease and a reduction of mineral fertilizer does not always lead to reduced N loadings because the agricultural systems can buffer even a slight surplus (Paper III). There is inertia in the catchments which can be seen in for example in the northern part of the catchment. Here atmospheric N deposition is almost as high as in the southern part but the nitrogen flux from these rivers is not elevated. These northern river catchments have N exports of the same magnitude as the natural background (Paper IV), indicating that the atmospheric N deposition is retained in the system and probably taken up by N limited boreal forests. However, important reductions can be achieved in the agricultural sector by detailed management of the planted land and animal manure. The highest sensitivity is in catchments with high animal density and high specific discharge, primarily draining to Kattegat and Danish Straits (Paper II & IV).
At the time of doctoral dissertation the following publications were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript

This investigation tested the hypothesis that iron, as a micronutrient, will affect proteins in Trichodesmium and therefore affect nitrogen fixation. Changes in proteins that are a result of iron enrichment were compared to naturally occuring diel changes. Alterations in the iron protein of nitrogenase were compared to nitrogen fixation rates using the acetylene reduction technique. The observed changes in proteins were compared in Trichodesmium colonies from the Caribbean Sea and the Sargasso Sea. Trichodesmium colonies were monitored for protein and iron content over a diel period on two cruises. The changes in protein and iron content in Trichodesmium colonies were variable but at times showed a cyclic diel pattern. Changes in protein bands on SDS-PAGE showed consistent changes in the banding pattern of a low molecular weight protein that responded to iron nutrition and time of day (Elardo and Rueter 1990). These changes were similar to changes m the iron protein of nitrogenase which also responded to changes associated with iron nutrition and time of day (Elardo 1991 ). Trichodesmium appear to alter certain proteins which appear as changes in banding patterns in response to environmental factors such as nutrients, temperature and light. My research shows that the pattern of modification of the iron protein of nitrogenase differs in colonies from the Caribbean Sea compared to those from the Sargasso Sea (Elardo 1991). The Caribbean Sea population in February had a clear pattern of active and inactive forms (day vs. night) of the enzyme. The Sargasso Sea population of Trichodesmium spp. had both forms of the enzyme at all times of the day during April and May when NO3 - is present in the euphotic zone due to recent mixing. These differences between the two populations may be due to different environmental conditions since the Caribbean Sea is permanently stratified, warmer and nutrient-depleted throughout the year. The Sargasso Sea undergoes seasonal breakdown of the thermocline during winter months, resulting in an injection of nitrate from deeper water, and minimum temperatures of 18oC.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently emerged as a novel intracellular calcium mobilising messenger in a variety of cells. Whilst increasing evidence suggests that NAADP acts on a distinct binding protein, little is known regarding the biochemical properties of the putative NAADP "receptor". My thesis investigates properties of the NAADP-binding protein in sea urchin eggs. Firstly, I show that NAADP binding to its target protein is inhibited by altering the protein:lipid ratio of soluble sea urchin egg homogenates - an effect prevented and reversed specifically by addition of exogenous phospholipids. These data highlight the importance of the lipid environment in maintenance of NAADP binding to its target protein. In addition, I show that upon binding its ligand, the NAADP-binding protein undergoes an unusual stabilization process that is dependent upon the time the receptor is exposed to its ligand. This property endows the NAADP-binding protein with the extraordinary ability to detect the duration of its activation. Finally I describe the development of a highly sensitive radioreceptor assay (based upon the sea urchin egg NAADP-binding protein) that is capable of detecting low levels of NAADP from cellular extracts. I apply this technique to determine NAADP levels in a variety of extracts prepared from cells under resting and stimulated conditions.

The upstream regulatory regions of numerous genes contain contiguous deoxyguanosine residues (G·C-rich sequences) which have been implicated in the regulation of gene expression, since they may involve alterations in their DNA structure, the binding of G-string factors and in some cases even the displacement of a nucleosome positioned over this area. A poly( dG).poly( de)-binding protein (suGF1) has previously been identified and purified on a small scale from embryonic nuclear extracts of the sea urchin Parechinus angulosus (1, 2). suGF1 binds in vitro to the H1-H4 intergenic region of the early histone gene battery, and the recognition site contains 11 contiguous Gs which are incorporated into a positioned nucleosome core in vitro. suGFI may be a member of a family of Gstring factors which could be involved in the developmental regulation of unrelated genes in various organisms. Prior to the commencement of this project no protein or DNA sequence information was available on the protein. The main objective of this thesis was to obtain the eDNA and the primary amino acid sequence for suGFI. Using this information, additional aims were to determine the developmental distribution of the protein and obtain insight into the molecular basis of the regulatory function of suGF 1 by analysis of the primary protein structure and expression of the eDNA.

The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.

La Serum Amiloide A3 (M‐SAA3) mamària és una proteïna de fase aguda expressada principalment a la glàndula mamària. Els nivells d’expressió de la M‐SAA3 varia en diferents situacions fisiològiques, el que suggereix un rol important a nivell funcional. Per tal d’analitzar les propietats de la proteïna, es van dur a terme quatre estudis. En el primer, la M‐SAA3 va ser expressada de forma recombinant en un sistema bacterià. Aquest pas és important ja que ens proporciona una font de proteïna, en casos en que la purificació de fonts naturals representa clarament un coll d’ampolla. Es va obtenir la seqüència de dues isoformes, però només una va ser expressada recombinantment. La principal diferència entre les dues formes era una deleció en la regió del motiu SNARE, el que suggeria que aquest motiu pot estar implicat en una activitat antibacteriana directe. A més, en el primer estudi es va analitzar la primera funcionalitat. La M‐SAA3 activava la fagocitosi mediada per macròfags, incrementant el nombre de macròfags actiu i la seva capacitat fagocítica. En el segon estudi es va analitzar l’efecte protector a nivell gastrointestinal. La M‐SAA3 clarament reduïa la translocació de bacteris enteropatògens en cèl∙lules CaCo‐2, una línia d’epiteli intestinal comercial. La M‐SAA3 també afectava la expressió de MUC3 i IL‐8, incrementant els seus nivells, el que connecta de forma directa la proteïna amb la resposta immune innata. En el tercer estudi, es va desenvolupar un model intestinal en boví a partir de cultius ex vivo de Plaques de Peyer. Aquests cultius ex vivo ofereixen un ambient únic on coexisteixen diferents tipus cel∙lulars i una aproximació més realista a la situació in vivo. En aquest context la infecció també va ser disminuïda i la M‐SAA3 incrementava els nivells de IL‐8 i INFγ. Per contra, les mucines no es van veure afectades, i la protecció va ser assolida per sobre‐expressió de Occludina i Claudina‐ 2, proteïnes que formen les tight junctions, encarregades de segellar la barrera epitelial. A més, es va demostrar que la M‐SAA3 activa cèl∙lules dendrítiques, incrementant l’expressió de citoquines i marcadors de maduració, migració i presentació d’antigen. En el quart i últim estudi, es va avaluar la possible aplicabilitat a nivell de la industria lletera. La M‐SAA3 va ser infosa intra‐mamàriament durant el secat, un període on es deixa de munyir les vaques per tal d’afavorir la regeneració cel∙lular i augmentar la seva productivitat. La M‐SAA3 va incrementar paràmetres relacionats amb una activació de l’involució tals com les metaloproteinases. Els nivells de proteïna i greix també eren augmentats i es va observar un augment numèric del recompte de cèl∙lules somàtiques. També es va observar que la proteïna incrementava l’expressió de IL‐8 i TNFα en cultius primaris de glàndula mamària, i també inhibia la infecció bacteriana. Finalment, les cèl∙lules dendrítiques també eren activades en absència d’infecció
Mammary Serum Amyloid A3 (M‐SAA3) is an acute phase protein mainly expressed in the mammary gland. The levels of the protein vary in different physiological situations, indicating that may play an important functional role. In order to analyze the protein properties four studies were performed. In the first study, the protein was recombinantly produced in a bacterial expression system. This was important, as difficulty in protein purification from natural sources is a clear bottleneck for functional studies. Two M‐SAA3 isoforms were obtained, but only one succeeded in the recombinant expression. Interestingly, the main difference was in a 3 amino acid deletion in the SNARE motif, which could be implicated in the direct bacterial killing. Moreover, the first functional role was evaluated. M‐SAA3 clearly enhanced macrophages phagocytosis, increasing both the number of active macrophages and the phagocytic capacity. In the second study, the protective effect at a gastrointestinal level was assessed. M‐SAA3 protein inhibited the translocation of enteropathogenic bacteria in CaCo‐2 cells, a commercial intestinal epithelial cell line. In addition, M‐SAA3 protein increased the expression of MUC3 and IL‐8, which directly connected the protein with the innate immune response activation. In the third study, a bovine intestinal model was developed using ex vivo Peyer’s Patches cultures. The ex vivo methodology offered a unique environment where different cell types coexist, and indeed, represent a more similar approach to an in vivo situation. In this context, the infection was also prevented, and a clear innate immune response was activated. M‐SAA3 clearly activated the expression of IL‐8, INFγ but in this case, mucins were not up‐regulated. The bacterial translocation was achieved by an increase in the Occludin and Claudin‐2 expression, tight junction genes that directly participate in the sealing of the epithelial barrier. Furthermore, the M‐SAA3 directly activated dendritic cells functions, increasing their cytokine expression profile and cellular markers related to maturation, migration and antigen presentation. In the fourth and last study, the potential applicability in dairy industry was evaluated. M‐SAA3 was infused in the mammary gland at dry off, a period of milking cessation which permits the mammary gland regeneration for an optimal production in following lactations. M‐SAA3 increased parameters related to an increased involution of the mammary gland, such as metalloproteinases. Also protein and fat were increased and a numerical increase in the somatic cell count was observed. In addition, M‐SAA3 raised the IL‐8 and TNFα levels in primary mammary gland cultures, and inhibited bacterial infection. Finally, dendritic cells were also activated by M‐SAA3 in absence of infection.

Group A streptococcus (GAS) is a strictly human pathogen that causes infections ranging from asymptomatic carriage to the highly lethal streptococcal toxic shock syndrome (STSS). GAS are classified according to the sequence of the variable 5’ end of the emm-gene that encodes the surface associated M-protein. In the late 1980s, outbreaks of GAS infections with high rates of STSS were reported in several parts of the world, including Sweden. Superantigens (SAgs), a group of exotoxins, have been described as key mediators of STSS due to their capacity to polyclonally activate T-cells and induce a massive release of inflammatory cytokines. Previous reports have revealed that sera from STSS patients have lower capacity to neutralize this SAg-mediated immune stimulation and a higher prevalence of GAS isolates with specific emm-genotypes during disease outbreaks. The aims of this thesis were to analyse the protective antibody response mounted by the host against SAgs produced by the infecting GAS isolate and to characterise the isolates emm-genotypes and SAg gene profiles. The clinical material examined was collected from patients with STSS, sepsis, erysipelas, or tonsillitis in Sweden between 1986 and 2001. Both acute- and convalescence-phase sera were analyzed, along with the infecting GAS isolates. The 92 clinical GAS isolates examined were found to exhibit a high degree of genetic diversity in terms of the number and identity of their SAg genes. Isolates with a given emm-genotype could be divided into subgroups on the basis of their SAg gene profiles. Ten different SAg gene profiles were identified in the 45 emm1 isolates examined; one of these ten was highly persistent, being observed in 22 isolates collected over 14 years. Two of the 11 known SAg genes in GAS, smeZ-1 and speA, were more prevalent in the emm1 associated profiles than in the SAg gene profiles of isolates with other emm-genotypes. Patients infected by GAS with the emm1-genotype were less likely to produce acute-phase sera that could effectively neutralize the T-cell mitogenicity induced by the infecting isolate’s extracellular products (EP). Sepsis patients whose sera exhibited this lack of neutralizing ability were more prone to developing STSS. Most patients whose acute-phase sera did not effectively neutralize the EP from the infecting isolate lacked protective antibodies in their convalescent-phase sera despite having elevated ELISA titers. The results reported herein show that combining SAg gene profiling with emm-genotyping may be useful for tracking the spread of GAS clones in the community. It was also shown that a lack of neutralizing activity in convalescence-phase sera might be due to an inability of those patients to mount a protective immune response against SAgs produced by the infecting GAS isolate.

This thesis describes structural studies on the interactions between the fucosylated chondroitin sulfate (fCS) oligosaccharides and human proteins known as selectins. fCS is a carbohydrate obtained from sea cucumbers, that can be classified as a branched glycosaminoglycan (GAG). It has attracted much attention due to its anti-coagulant, anti-inflammatory, antimetastatic and anti-HIV properties and its structure was previously determined by NMR. Selectins constitute a family of proteins involved in cell adhesion processes, such as inflammation, attachment of viral particles and migration of tumour cells. fCS oligosaccharides have been shown to bind to selectins, which is likely a reason behind their biological activity. However, the mechanism of this interaction is currently unknown. The initial part of the thesis describes the experimental work on expression and purification of the recombinant L- and P-selectin constructs in Pichia pastoris, Escherichia coli and HEK 293 cells. The aim of these experiments was to produce two constructs for each selectin, a single domain construct, consisting of the C-type lectin domain only, and a double domain construct, consisting of both the C-type lectin and the EGF-like domains. The intention was that the recombinant proteins would be labelled with 13C and 15N to allow for the in-depth structural NMR studies on the fCS-selectin interaction. Various experimental approaches have been explored, including the use of different cell lines, modifications to construct design, as well as alterations to expression and purification conditions. Although it was not possible to produce soluble selectin constructs in either bacterial or yeast cells, protein expression tests in HEK293 cells, performed in collaboration with the Oxford Protein Production facility (OPPF), led to production of a soluble L-selectin construct, consisting of the L-selectin C-type lectin domain. The produced L-selectin construct, as well as two commercially available constructs of the Land P-selectin extracellular domains, were used in the Saturation Transfer Difference (STD) NMR experiments to provide new information about the nature of the fCS-selectin binding. The STD experiments allowed to identify the regions within the fCS oligosaccharides that are in direct contact with the protein and likely play an important role in this interaction. Experiments on different protein constructs allowed the comparison of fCS binding to P-selectin and to two different recombinant constructs of L-selectin. Results of these studies suggest that the binding occurs via a similar mechanism for both L- and P-selectins and that the fCS oligosaccharides bind to one-domain L-selectin construct with similar affinity as to a larger construct, consisting of the entire extracellular region of the protein. Alongside the experimental work, theoretical in silico studies on the fCS-selectin binding were undertaken as part of this project. The existing X-ray structures of selectin complexes were subjected to Molecular Dynamics (MD) simulations, which allowed to explore the dynamic behaviour of E-selectin upon binding to sialyl Lewis x (sLex). It was found that sLex forms a more favourable interaction with the extended conformation of E-selectin and that the protein in this conformation is characterised by a high degree of interdomain flexibility, with a new type of interdomain movement observed in the MD studies on this complex. In further in silico studies, the fCS oligosaccharides were docked to the existing P-selectin structures. The docking tests were performed on the computationally produced fCS trisaccharides with fucose branches either 2,4 or 3,4-sulfated. Results were evaluated with MD simulations and analysed in the light of current knowledge of selectin-ligand binding and the STD NMR experimental results. The in silico studies allowed to identify a subset of P-selectin residues that are likely involved in the interaction with fCS oligosaccharides in vivo. The conformational behaviour of P-selectin upon binding to fCS was also explored and it was found that the interdomain hinge is flexible during this interaction and allows transition from bent to extended conformational state. Finally, a new NMR method was developed to facilitate the studies of complex carbohydrates, incorporating the concepts of G-matrix Fourier Transform (GFT) NMR into 2D HSQC and 2D HSQC-TOCSY experiments. The method allows to separate peaks in the regions of high spectral overlap, providing information that can simplify the assignment process. The new experiments facilitated the structural evaluation of a sample containing a mixture of oligosaccharides resulting from the depolymerisation of fCS polysaccharide.

Visando novos estudos com a proteína amilóide sérica A (SAA), propusemos a produção de seu recombinante humano em Escherichia coli, mais especificamente, a isoforma encontrada na fase aguda (A-SAA1) e da constitutivamente expressa (C-SAA4). Realizamos a expressão, identificação e purificação das proteínas recombinantes. Concomitantemente, também avaliamos o efeito da hipóxia na expressão e produção da proteína SAA nativa em linhagens de pré-adipócitos murinos 3T3-L1, não diferenciados e diferenciados e adipócitos humanos. Aparentemente quanto maior o grau de diferenciação celular, maior a expressão e produção da proteína. Para os adipócitos humanos, o perfil de expressão de mRNA da SAA mostra que SAA1>SAA2>SAA4 nas relações 500:150:1. Na hipóxia, há um aumento na expressão de SAA, entretanto não associamos esta expressão a um aumento da concentração da proteína. A importância do reconhecimento de que SAA pode ser umas das proteínas induzidas pela hipóxia em adipócitos é discutida em relação ao seu papel pró-inflamatório.
In order to provide more complex studies with the protein serum amyloid A (SAA), this study proposed the production of its human recombinant in bacteria (Escherichia coli), more specifically, the synthesis of the main isoform found in the acute phase (A-SAA1) and the constitutively expressed (C-SAA4). The expression, identification and purification of the recombinants proteins was performed. Concurrently, we also did a study evaluating the effect of hypoxia in the expression and protein production of native SAA in undifferentiated and differentiated murine preadipocytes 3T3-L1 and humans adipocytes. Apparently, the protein expression and production increases with the cell differentiation degree. For human adipocytes, we demonstrated the mRNA expression profile of SAA, in which SAA1 > SAA2 > SAA4 (500:150:1). In hypoxia, there is an increased expression of SAA, but we could not link it to an increase of protein concentration. The importance of recognizing that SAA may be one of the proteins induced by hypoxia in adipocytes is discussed in relation to the proinflammatory role of this protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is arguably the most economically devastating pathogen affecting the global swine industry. Since the emergence of the virus in the late 1980s, vaccination strategies aimed to control the virus have not been very effective. Current commercial vaccines are generally protective against homologous or closely-related strains but ineffective at conferring heterologous protection against genetically-diverse strains of the virus. Consequently, emergence of variant and sometime more pathogenic strains of PRRSV continues in global swine herds. As such, there is a need for better understanding of the molecular mechanisms involved in the replication of the virus. In order to better understand the molecular mechanisms of host responses to PRRSV replication, we first sought to evaluate the ability of the virus to induce stress granules (SGs) during PRRSV infection. SGs are intracellular, cytoplasmic aggregates of RNA-binding proteins (RBPs) and mRNA. Formation of SGs is observed upon cellular stress and ultimately function to arrest cellular translation to promote cellular survival until the stress has been remedied. Indeed, several viruses have been shown to modulate the SG pathways to facilitate viral replication and even suppress the host's immune response. However, it is currently unknown whether PRRSV modulates the SG response. First, we used confocal microscopy and fluorescent in situ hybridization (FISH) to determine the distribution of known SG marker proteins and cellular mRNAs. Our findings revealed that PRRSV induces a potent SG response at late time points post-infection, and that SGs were closely associated with viral replication complexes (VRCs). Subsequently, we demonstrated that SGs are dispensable for viral replication, as short hairpin RNA (shRNA)-mediated knockdown of critical SG components (G3BP1 and G3BP2) did not affect viral replication. Interestingly, we found that the PRRSV-induced SGs are formed in a PERK-dependent manner. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Further investigation into the PERK signaling pathway revealed that PRRSV induces a significant amount of ER stress upon the cell during viral infection, and that exogenous stress significantly impaired the ability of the virus to replicate in MARC145 cells. We also showed that PRRSV potently induces all three signaling branches of the UPR, including PERK. While PERK knockdown had no effect on cell viability or viral replication, it significantly upregulated the mRNA expression of interferon-β and interferon stimulated genes (ISGs). The results from our studies suggest a critical role for PERK in regulating the host innate immune response to PRRSV infection. Only with a better understanding of the underlying molecular mechanisms of PRRSV replication will we be able to rationally design more effective vaccines against the virus.
Doctor of Philosophy
Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically-devastating disease in the global swine industry. Annually, PRRSV is estimated to cause more than $600 million in economic losses to the swine industry in the United States alone. Current commercial vaccines against the virus are not effective against the diverse field strains largely due to the extreme heterogeneity of the virus. PRRSV is also able to potently suppress several aspects of the host's immune response and therefore establish a persistent infection. The underlying mechanisms of PRRSV-mediated immune suppression are not well understood. Therefore, in this dissertation we decided to investigate the molecular mechanisms of host responses to PRRSV infection. We first investigated the ability of the virus to induce stress granules (SGs). SGs are important intracellular regulatory components that modulate many aspects of the host's cellular processes, and have even been shown to play roles in regulating viral replication and controlling immune responses to viral infection. We demonstrate that PRRSV not only induces SGs, but that the PRRSV-induced SGs are closely associated with viral replication complexes (VRCs) within infected cells. The PRRSV-induced SGs were dispensable for viral replication. PRRSV-induced SGs were previously shown to form in a PERK dependent manner. Therefore, in the second part of this dissertation research, we decided to investigate the PERK signaling pathway during PRRSV infection. PERK is an important sensor of ER stress and activator of the unfolded protein response (UPR). Our results showed that PRRSV potently induces ER stress and all three signaling branches of the UPR, including PERK. Furthermore, we revealed that PERK may play an important role in regulating the type I interferon response to PRRSV infection. The results from our studies will aid in understanding the underlying molecular mechanism of PRRSV replication which will help rationally design the next generation of more effective vaccines against this devastating swine pathogen.

Thesis advisor: David R. Burgess
Polarity established by the first cleavages in sea urchin embryos was investigated in this thesis revealing precocious embryonic polarity. Studies of embryonic polarity have focused on protostomes such as C. elegans, and those on deuterostomes have focused on later developmental stages. I find asymmetries in the sea urchin membrane cell cortex as early as the first division after fertilization as a result of new membrane addition in the cleavage furrow. Membrane domains and the polarity determinants Par6, aPKC, and Cdc42 are polarized to the apical, or free, cell surface, while the cell-cell contact site remains distinct. Using immunofluorescence, fluorescence recovery after photobleaching (FRAP), and specific inhibitor treatments, myosin filaments were identified as the major regulator of membrane cortex polarity. However, membrane domains and cortical polarity determinants are differentially regulated with respect to blastomere dissociation. These asymmetries are required for proper spindle alignment and cleavage plane determination and are responsible for polarized fluid phase endocytosis. The work in this thesis and future studies addressing the connection between the membrane cortex and myosin filaments has and will lead to a greater understanding of the maintenance of embryonic polarity in cleavage stage sea urchin embryos
Thesis (PhD) — Boston College, 2009
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

L’atròfia muscular espinal (AME) és una malaltia neuromuscular causada per mutació o deleció en el gen SMN1, que codifica per la proteïna ubiqua SMN (de l’anglès survival motor neuron). L’AME es caracteritza per atròfia muscular i degeneració de les motoneurones (MN) de la medul·la espinal. Els esdeveniments moleculars que causen la vulnerabilitat específica de les MN amb nivells baixos de proteïna SMN encara no es coneixen. La via de l’NF-κB (nuclear factor-κB) ha destacat recentment ja que sembla jugar un paper cabdal en la supervivència de les MN i en les malalties neurodegeneratives. Els factors de transcripció NF-κB regulen gens relacionats amb molts processos cel·lulars. En aquest treball hem analitzat la capacitat dels membres de la via de l’NF-κB de regular la proteïna Smn i el seu possible rol en la patogènesi de l’AME. L’activació de la via de l’NF-κB està associada a la fosforilació de l’IKKα/IKKβ i la translocació nuclear del factor RelA/p50 (via canònica) o la fosforilació de l’homodímer d’IKKα i la translocació nuclear del factor RelB/p52 (via no canònica). La inhibició de diferents membres d’aquestes vies (tant la canònica com la no canònica) usant la metodologia de transducció amb lentivirus amb shRNA en cultius primaris de MN embrionàries aïllades de ratolí hem demostrat que una reducció selectiva del factor RelA provoca una reducció de la proteïna Smn, mentre que una reducció del factor RelB no té cap efecte en els nivells de l’Smn. En el nostre model cel·lular, la reducció dels nivells de l’IKKα o l’IKKβ provoca un efecte oposat en l’Smn. Mitjançant la tècnica de la RT-PCR hem observat que la transducció de les MN amb l’shIKKβ provoca un augment dels nivells de l’mRNA de Smn, mentre que la transducció amb l’shIKKα o l’shRelA no modifica l’expressió de Smn. El doble knockdown de l’IKKα i l’IKKβ a les MN mostra una reducció de l’Smn. El knockdown selectiu de l’IKKα o l’IKKβ presenta una reducció de la fosforilació del RelA, es coneix que aquesta fosforilació en permet l’alliberament del seu inhibidor al citosol i en facilita la translocació nuclear. També la proteïna CREB, un dels factors de transcripció coneguts de l’Smn, disminueix amb la transducció de les MN amb els shIKKα o amb l’IKKα i l’IKKβ alhora, així com amb l’shRelA. Ara bé, les motoneurones amb l’shIKKβ mostren una reducció de la fosforilació de RelA però un augment dels nivells de CREB. La transducció de les MN amb l’shCREB disminueix els nivells de l’Smn recolzant el paper regulador de CREB en l’Smn. Hem observat una reducció de l’IKKα, l’IKKβ i de la fosforilació de RelA amb la transducció de les MN amb l’shSmn i en les MN del model murí sever de l’AME. Els nostres resultats mostren l’habilitat de la via canònica de l’NF-κB de regular els nivells de l’Smn i que aquesta via també es troba alterada en les MN deficients en la proteïna Smn. En conjunt, aquestes observacions suggereixen que la via de l’NF-κB pot tenir un rol en la patogènesi i ser, a la vegada, una possible diana terapèutica per l’AME.
La Atrofia Muscular Espinal (SMA) es un trastorno neuromuscular causado por la mutación o deleción del gen SMN1, el cual codifica para la proteína que se expresa ubicuamente SMN (del inglés Survival Motor Neuron). La AME se caracteriza por atrofia muscular y degeneración de las motoneuronas de la médula espinal (MN). Los eventos moleculares detrás de la vulnerabilidad selectiva de las MN con niveles bajos de la proteína SMN se desconocen. La vía del factor nuclear-kB (NF-kB) ha sido implicada recientemente en la supervivencia de las MNs, así como en trastornos neurodegenerativos. Los factores de transcripción NF-kB regulan genes relacionados con varios procesos celulares. En este trabajo hemos analizado la capacidad de los miembros de la vía del NF-κB de regular la proteína SMN y su posible rol en la patogénesis del AME. La activación de la vía del NF-κB está asociada a la fosforilación de IKKα / IKKβ y la translocación nuclear del factor RelA/ p50 (vía canónica) o la fosforilación del homodímero de IKKα y la translocación nuclear del factor RelB / p52 (vía no canónica). Hemos realizado la inhibición de diferentes miembros de estas vías (tanto la canónica como la no canónica) usando la metodología de shRNA, y la transducción mediante el uso de lentivirus, en cultivos primarios de MN embrionarias aisladas de ratón. Hemos demostrado que una reducción selectiva del factor RelA provoca una reducción de la proteína SMN, mientras que una reducción del factor RelB no tiene ningún efecto en los niveles de la SMN. En nuestro modelo celular, la reducción de las proteínas IKKα o IKKβ mostró efectos opuestos sobre la proteína Smn. Mediante la técnica de PCR, hemos observado que la transducción de las MN con el shIKKβ provoca un aumento de los niveles de mRNA de SMN, mientras que la transducción con el shIKKα o el shRelA no cambian los niveles de RNA de SMN. El doble knockdown de IKKα e IKKβ en las MN muestra una reducción de SMN. El knockdown selectivo de IKKα o IKKβ presenta una reducción de la fosforilación del RelA, esta fosforilación permite la liberación de su inhibidor en el citosol y facilita la translocación nuclear. La proteína CREB, uno de los factores de transcripción conocidos para SMN, disminuye con la transducción de las MN con shIKKα o con IKKα e IKKβ a la vez, así como con shRelA. Ahora bien, las motoneuronas transducidas con shIKKβ muestran una reducción de la fosforilación de RelA pero un aumento de los niveles de la proteína CREB. La transducción de las MN con el shCREB disminuyó los niveles de la proteína SMN apoyando el papel regulador de CREB sobre SMN.
Spinal Muscular Atrophy (SMA) is a neuromuscular disorder caused by mutation or loss in SMN1 gene, encoding the ubiquitously expressed Survival Motor Neuron (SMN) protein. SMA is characterized by muscle atrophy, and spinal cord motoneurons (MNs) degeneration. The molecular events behind the selective vulnerability of these MNs to low level of SMN protein are still unknown. The nuclear factor-κB (NF-κB) pathway has recently been emerged having a vital role related to MN survival, and in neurodegenerative disorders. The NF-κB transcription factors regulate genes related to several cellular processes. In the present work, we have analyzed the ability of NF-κB pathway members to regulate Smn and their possible role in SMA pathogenesis. The NF-κB pathway activation is associated with IKKα/IKKβ phosphorylation, and RelA/p50 nuclear translocation (canonical) or IKKα homodimer phosphorylation, and RelB/p52 nuclear translocation (non-canonical). The inhibition of different protein members of both canonical, and non-canonical pathways using shRNA lentiviral transduction methodology in a primary culture of isolated embryonic spinal cord MNs reveals that the selective reduction of RelA induced the reduction of Smn whereas RelB protein reduction had no effect on Smn. In our culture system, reduction of IKKα or IKKβ proteins showed opposite effects on Smn. RT-PCR studies indicate that the shIKKβ-transduced MNs showed increased Smn mRNA levels, whereas it was not observed changes in Smn mRNA in the case of shIKKα- or shRelA-transduced MNs. The double knock-down of IKKα and IKKβ in MNs showed Smn reduction. The knockdown of IKKα and/or IKKβ showed a decrease in RelA phosphorylation, where the phosphorylation of RelA enable RelA/p50 release from its inhibitor in the cytoplasm and facilitates their nuclear translocation. Also, the CREB, one of the transcription factors for Smn was decreased in shIKKα, or in shIKKα- plus IKKβ-transduced MNs, and as well as in shRelA-transduced MNs. But, the shIKKβ MNs exhibited reduced p-RelA but increased CREB level. The shCREB-transduced MNs decreased Smn level, authenticating the regulatory role of CREB on Smn. We observed a reduction in IKKα, IKKβ and p-RelA levels in shSmn-tranduced MNs, and in MNs from a severe type SMA mouse model. Our results show the ability of NF-κB canonical pathway to regulate Smn level and, conversely, this pathway is also altered in Smn-deficient MNs. Together, these observations suggest that the NF-κB pathway has a role in SMA pathogenesis, and could be a therapeutic target for SMA.

Sílicas mesoporosas, como a SBA-15, constituem-se de partículas de óxido de silício que, devido às suas propriedades físico-químicas apresentam potencial adjuvante. Para entender os mecanismos de ação da SBA-15, camundongos foram imunizados, pelas vias oral e/ou subcutânea (s.c.), com a proteína recombinante HBsAg ou ovalbumina (OVA) e apresentaram aumento significativo nos títulos de anticorpos específicos após imunização s.c. com ambos os antígenos em sílica; entretanto, somente a administração de HBsAg: SBA-15 induziu a produção de anticorpos pela via oral. O efeito da sílica na ativação de células dendríticas foi verificado após incubação com diferentes concentrações de SBA-15, indicando aumento na produção de IL-6, na proliferação e produção de IFN-g por linfócitos T após a apresentação de OVA ou seus peptídeos. A resposta de linfócitos T in vivo mostrou aumento na resposta imune celular de animais que receberam a sílica, mas não indicou a indução da resposta de linfócitos T citotóxicos. Esses resultados confirmam o efeito adjuvante da SBA-15, principalmente para a resposta de anticorpos, e indicam que a sílica pode aumentar a disponibilidade dos antígenos às APC, auxiliando na apresentação antigênica.
Amorphous silicon oxide particles named SBA-15 are promising adjuvant vectors due to its physicochemical properties and the aim of this study is to explore how they might act in promoting immune responses. Mice were orally and/or subcutaneously (s.c) immunised with the recombinant protein HBsAg or ovalbumin (OVA), showing a significant increase in the antibody titers after s.c. immunisation with both antigens in silica; however, only the administration of HBsAg: SBA-15 induced antibody production after oral immunisations. The activation of dendritic cell by silica was assessed by pulsing those cells with different concentrations of the particles, suggesting the interference of SBA-15 in the production of IL -6 and in T cell proliferation as well as IFN-g production by T lymphocytes after presentation of this protein or its peptides in vitro SBA-15 was able to enhance T cell responses in vivo but did not allow OVA to induce specific cytotoxic T lymphocyte activity. These preliminary data confirm that SBA-15 acts as an adjuvant for antibody responses and suggest that its effects may reflect enhanced availability of antigen, rather than direct effects on antigen presenting cells such as DC.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of the lower motor neurons. It is caused by homozygous loss or mutations in the Survival Motor Neuron 1 (SMN1) gene, leading to a reduction of the amount of Survival Motor Neuron (SMN) protein. In human patients, an almost identical copy gene, SMN2, is unable to fully compensate for the lack of SMN1, due to a C to T transition in exon 7. Despite progress in the field, the cellular site of action of the SMN protein remains incomplete. Fully defining it is critical for clinical treatments and to enhance our understading of the basic biology of the disease. These questions have been addressed in large part through the study of SMA model organisms. These studies have shown that SMN is particularly important in motor neurons; additional experiments have explored various other tissues which may also be particularly sensitive to SMN, including skeletal muscle. Still, the role of muscle in SMA continues to be debated and the precise function of SMN in this tissue as it pertains to the pathology of the disease remain far from clear. This is partly because in vivo experiments performed to date cannot rule out the possibility that changes observed in muscle are simply occurring as a secondary effect of denervation due to pathology in the innervating motor neurons. As therapies for SMA evolve, these remain important questions to address. Accordingly, the goal of this research project has been to more precisely delineate the contributing role of muscle in the overall pathology/phenotype characteristic of SMA. Our results show that wild-type levels of SMN in muscle are absolutely critical to the maintenance of healthy muscle. Thus we believe that SMN functions cell-autonomously within this tissue to ensure its health and viability. Restoring protein to muscle is therefore expected to constitute a vital aspect of treating SMA in SMN repletion-type therapies.
La Atrofia Muscular Espinal (AME) es un trastorno neuromuscular autosómico recesivo caracterizado por la degeneración de las neuronas motoras inferiores. Es causada por pérdida homocigótica o mutaciones en el gen de la neurona motora de supervivencia (SMN1), que conduce a una reducción de la cantidad de proteína de neurona motor de supervivencia (SMN). En los pacientes humanos, un gen de copia casi idéntico, SMN2, es incapaz de compensar completamente la falta de SMN1, debido a una transición de C a T en el exón 7. A pesar del progreso en el campo, el sitio celular de acción de la proteína SMN permanece incompleto. Definirlo completamente es crítico para los tratamientos clínicos y para mejorar nuestro conocimiento de la biología básica de la enfermedad. Estas preguntas se han abordado en gran parte a través del estudio de organismos modelo AME. Estos estudios han demostrado que la SMN es particularmente importante en las neuronas motoras; experimentos adicionales han explorado otros tejidos que también pueden ser particularmente sensibles a SMN, incluyendo el músculo esquelético. Sin embargo, el papel del músculo en SMA sigue siendo debatido y la función precisa de SMN en este tejido en lo que respecta a la patología de la enfermedad permanecen lejos de ser clara. Esto se debe en parte a que los experimentos in vivo realizados hasta la fecha no pueden descartar la posibilidad de que los cambios observados en el músculo estén simplemente ocurriendo como un efecto secundario de la denervación debido a la patología en las neuronas motoras innervantes. A medida que evolucionan las terapias para AME, éstas siguen siendo importantes cuestiones a responder. En consecuencia, el objetivo de este proyecto de investigación ha sido delimitar con mayor precisión el papel contributivo del músculo en la característica general de patología/fenotipo de AME. Nuestros resultados muestran que los niveles en estado natural de SMN en el músculo son absolutamente críticos para el mantenimiento del músculo sano. Por lo tanto, creemos que SMN funciones de células autónomas dentro de este tejido para garantizar su salud y viabilidad. Por lo tanto, se espera que la restauración de la proteína en el músculo constituya un aspecto vital del tratamiento de AME en las terapias de repleción de SMN.

Thesis advisor: David Burgess
This doctoral thesis addresses two central topics divided into separate chapters. In Chapter 1: The cortical response to RhoA is regulated during mitosis, experimental findings using sea urchin embryos are presented that demonstrate that the small GTPase RhoA participates in positive signaling for cell division and that this activity is negatively regulated prior to anaphase. In a second series of experiments, myosin phosphatase is shown to be a central negative regulator of myosin activity during the cell cycle through metaphase of mitosis and experimental findings support the conclusion that myosin phosphatase opposes RhoA signaling until anaphase onset. These experiments also reveal that myosin activation alone is insufficient to stimulate cortical contractions during S phase and during metaphase arrest following activation of the spindle checkpoint. In Chapter 2: Annotation of cytoskeletal and motility proteins in the sea urchin genome assembly, as part of a collaborative project, homologs of cytoskeletal genes and gene families were derived and annotated from the sea urchin genome assembly. In addition, phylogenetic analysis of multiple gene families is presented based on these findings
Thesis (PhD) — Boston College, 2008
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology

During the past several decades, corals worldwide have been dealing with a considerable increase in water temperature due to climate change, which is predicted to increase the frequency of coral bleaching and mass mortality events. Nevertheless, corals show differences in stress susceptibility and they are not all affected evenly. The symbiotic coral Oculina patagonica from the Eastern Mediterranean Sea can thrive in relatively unstable environments and is considered a stress-tolerant species. In this study, baseline expression and temporal dynamics of induction of a 70-kDa heat shock protein (HSP70) after an acute heat stress were analyzed in O. patagonica to investigate the influence of its peculiar physiological traits on stress responsiveness. Furthermore, data collected were further discussed within a comparative analysis with similar findings reported in 5 temperate corals of the Mediterranean Sea (Franzellitti et al., 2018). Results show that O. patagonica hsp70 transcriptional response aligns with the formerly observed high resistance for elevated sea water temperatures of this species. The multispecies comparison shows that hsp70 expression varies in accordance with the stress sensitivity of coral populations inhabiting different thermal environments and possessing different trophic strategies and morphologies. This study also reports an analysis of the post heat-stress transcriptional regulation of transcripts related to energy metabolism (gadph), redox regulation (sod), and DNA damage (bcl-2 and bax), disclosing the time line of the events occurring in O. patagonica in response to an acute heat stress, which aligns with its quick recovery from bleaching. These molecular processes analysis is particularly demanding for corals inhabiting the Mediterranean Sea, in light of projected scenarios of anthropogenic global change.

Aunque el uso de altos niveles de fuentes de proteína vegetal en piensos para doradas de engorde se ha alcanzado con éxito en cuanto al crecimiento, estas dietas todavía están asociadas a efectos negativos en la eficiencia nutricional y en la capacidad inmunitaria. El intestino es el órgano donde se produce la primera interacción entre el pez, los nutrientes y las bacterias del medio, y desarrolla un papel crucial en la digestión de los nutrientes y la respuesta infamatoria e inmune. Esta tesis doctoral se centra en el impacto de distintas dietas con altos niveles de proteína vegetal, y especialmente, en la evaluación del estatus intestinal de las doradas de engorde alimentadas con altos niveles de sustitución de la harina de pescado durante un periodo largo de tiempo. Los cambios observados en el intestino se caracterizaron mediante el uso de distintas estrategias, como el análisis de la digestibilidad y la retención de amino ácidos, de la excreción de amonio, de la actividad de enzimas digestivos, de los cambios histológico o de la expresión de genes relacionados con la función y el mantenimiento de la arquitectura intestinal, así como técnicas ómicas para el análisis del proteoma y de la microbiota intestinal. Se ensayaron distintos niveles de sustitución de harina de pescado, pero el impacto de las dietas con una sustitución completa, bien complementada con subproductos de origen marino o suplementada con aminoácidos libres sintéticos, recibió mayor atención. La sustitución completa de la harina de pescado provocó una reducción, aunque ligera, del crecimiento y de la eficiencia digestiva y nutritiva de la dorada de engorde, aunque el impacto sobre el crecimiento era mayor cuando los peces eran alimentados desde la época de juveniles con estas dietas. La digestibilidad y el nivel de síntesis de proteína se vio alterada, aunque no se observaron diferencias significativas en la actividad enzimática digestiva. No obstante, el impacto de las fuentes vegetales cuando no había fuentes de proteína marina en la dieta era especialmente crítico para la supervivencia de los peces. En el intestino de estos peces solo se observaron diferencias menores relacionadas con la inflamación a nivel histológico, pero también se observó una disminución en la expresión génica de genes involucrados en la inflamación y la respuesta inmune. El análisis de la microbiota intestinal reveló cambios significativos en la composición de su composición, especialmente en el intestino posterior, sugiriendo una posible falta de capacidad de regular la respuesta inmune y de modular la colonización de bacterias patógenas tras un largo periodo de alimentación con esta dieta. Por otro lado, el análisis del proteoma de la mucosa intestinal también mostró un claro impacto sobre distintos procesos biológicos relacionados con el mantenimiento del homeostasis intestinal y de la integridad epitelial. Por el contrario, no se observó un impacto de la sustitución de la harina de pescado a nivel de expresión génica o del proteoma cuando se incorporaba a la dieta una fuente de proteína marina complementaria, aunque sí que se observaron algunos signos menores de inflamación. Por último, se desarrolló un sistema ex vivo para estudiar la respuesta inflamatoria e inmune de la mucosa intestinal a la presencia de distintas bacterias, y se realizó un ensayo preliminar en dorada para evaluar el efecto de la dieta sobre esta respuesta. En resumen, en este trabajo se ha realizado una evaluación extensa y detallada de los efectos a nivel intestinal de la inclusión de altos niveles de proteína vegetal en la dieta para doradas de engorde. Los resultados indican que las alteraciones en la capacidad inmune, la homeostasis y la microbiota intestinal aparecían solo cuando la proteína procedía exclusivamente de fuentes vegetales, y podrían explicar la mayor mortalidad registrada con esta dieta.
Malgrat que la utilització d'alts nivells de proteïna vegetal en pinsos per a dorades en la fase d'engreixament s'ha aconseguit amb èxit en quan al creixement, aquestes dietes encara s'associen amb freqüència amb efectes negatius en l'eficiència nutricional i la capacitat immunitària. L'intestí és l'òrgan on es produeix la primera interacció entre el peix, els nutrients de la dieta i les bactèries de l'ambient, i juga un paper fonamental en la digestió dels nutrients i en la resposta inflamatòria i immune. Aquesta tesi doctoral es centra en l'impacte de diferents dietes experimentals amb un alt nivell de proteïna vegetal, i especialment, en l'avaluació de l'estat de l'intestí de les dorades d'engreixament alimentades durant un llarg període amb alts nivells de substitució de farina de peix. Els distints canvis observats a nivell intestinal es van descriure mitjançant l'ús de distintes estratègies, com l'anàlisi de la digestibilitat i la retenció dels aminoàcids, de l'excreció d'amoni i de l'activitat enzimàtica, dels canvis histològic o de l'expressió de gens relacionats amb la funció i el manteniment de l'estructura intestinal, així com tècniques òmiques per a l'anàlisi del proteoma i de la microbiota intestinal. Es van assatjar diferents nivells de substitució de farina de peix, però l'impacte de les dietes amb substitució completa, bé complementada amb subproductes d'origen marí o suplementada amb aminoàcids lliures sintètics, va rebre major atenció. La substitució completa de la farina de peix va tenir un efecte lleugerament negatiu sobre el creixement i l'eficiència digestiva i nutritiva de la dorada d'engreixament, encara que l'impacte era major quan els peixos eren alimentats des de la fase de juvenils amb aquesta dieta. La digestibilitat i el nivell de síntesis de proteïna es va veure alterada, encara que no s'observaren diferències significatives en l'activitat dels enzims digestius. No obstant, l'impacte de les fonts vegetals quan s'eliminaven per complet les fonts de proteïna marina de la dieta era especialment crític en la supervivència dels peixos. En l'intestí d'aquests peixos sols s'observaren xicotets indicis d'inflamació a nivell histològic, però també es va observar una disminució l'expressió de gens involucrats amb el procés inflamatori i la resposta immune. L'estudi de la microbiota intestinal va revelar canvis significatius en la composició, especialment a l'intestí posterior, suggerint una possible falta de capacitat de regular la resposta immunitària i de modular la colonització per part de patògens després d'un llarg període d'alimentació amb aquesta dieta. D'altra banda, l'anàlisi del proteoma de la mucosa intestinal també va mostrar un impacte clar sobre diferents processos biològics relacionats amb el manteniment de l'homeòstasi intestinal i de la integritat de l'epiteli. Per contra, no es van observar un impacte de la substitució de la farina de peix a nivell d'expressió gènica o proteoma quan s'incloïa a la dieta una font complementària de proteïna d'origen marí, encara que sí que s'observaven alguns signes d'inflamació. Per últim, es va desenvolupar un sistema ex vivo per avaluar la resposta inflamatòria i immune de la mucosa intestinal davant la presència de diferents bactèries, i es va realitzar un assaig preliminar per determinar l'efecte de la dieta sobre aquesta resposta. En resum, en aquest treball s'ha realitzat una avaluació extensa i detallada dels efectes a nivell intestinal de la inclusió d'alts nivells de fonts de proteïna vegetal a les dieta per a les dorades d'engreixament. Els resultats indiquen que les alteracions en la capacitat immunitària, l'homeòstasi i la microbiota intestinal eren observades solament quan la proteïna era exclusivament obtinguda de fonts vegetals, i podrien explicar la major mortalitat observada amb aquesta dieta.
Although the inclusion of plant protein sources at high levels in aquafeeds for on-growing gilthead seabream has been successfully achieved on gilthead seabream in terms of growth, these diets are still associated to detrimental effects in feed efficiency and immune capacity. The intestine is the organ where takes place the first interaction of the host with dietary antigen or environmental bacteria, and plays a major role in the digestion of nutrients and the inflammatory and the immune response. The present PhD thesis focus on the impact of classical formulated high plant protein diets on fish performance, but especially, on evaluation of the intestinal status in on-growing fish long-term fed with high levels of fishmeal replacement. Changes at intestinal level were characterized by using different approaches, including protein and amino acid digestibility and retention and ammonia excretion, digestive enzyme activity, histology, expression of genes related with inflammation, immunity, structure and digestion, but also using whole tissue-level techniques for the analysis of the impact on proteome and gut microbiota. Different levels of fishmeal replacement were assayed, although the impact of diets with total replacement, complemented by inclusion of alternative marine by-products or supplemented by free amino acids, received greater attention. Total fish replacement produced a negative but minor impact on the growth and nutritive and digestive performance of on-growing gilthead seabream. Nevertheless, when fish were fed from juvenile stage with plant protein based diets, a higher negative impact in growth terms was noticed. Digestibility and metabolic use of amino acids was altered, but no differences were observed in the digestive enzyme activities. Nonetheless, feeding fish with total dietary fishmeal replacement by plant protein without any marine protein source was especially critical for survival rate. In these fish, gut histological assessment only revealed minor alterations related with an inflammatory response, but gene expression assay showed a down-regulation of several genes involved in the inflammatory and immune response. Moreover, a drastic change in the microbiota composition was observed, especially at the hindgut, revealing a possible lack of capacity to regulate a defensive response and to face with pathogen colonisation after a long-term coupling with these diet. Likewise, gut mucosa proteome analysis also suggests an impact on biological processes related with the maintenance of gut homeostasis and the epithelial integrity. In contrast, total fishmeal replacement did not induce alterations at transcript or proteomic level when diet was complemented with marine ingredients, although some minor inflammatory signs were reported. On the other hand, an ex vivo system to study the inflammatory and immune response of the gut mucosa to the presence of different bacteria was developed, and a preliminary assay evaluating the impact of the diet on this response was performed. To sum up, present works represents a wide assessment at intestinal level of the effects of including plant protein sources at high levels in aqua feeds for on-growing gilthead seabream. Results indicate that alterations in the immune capacity, the gut homeostasis and the microbiota were observed when protein was exclusively provided by plant sources, and could explain the higher mortality reported with this diet.
Estruch Cucarella, G. (2018). ASSESSMENT OF THE LONG-TERM IMPACT OF HIGH PLANT PROTEIN DIETS ON THE INTESTINAL STATUS OF THE ON-GROWING GILTHEAD SEA BREAM (Sparus aurata, L.) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/113063
TESIS

Due to heavy nutrient load and adverse climate change the occurrence of toxic cyanobacterial blooms have significantly increased during the last decades. Nodularia spumigena is one of the dominant toxic cyanobacteria which produces massive and inherent blooms in brackish water body, the Baltic Sea, particularly in late summer. Nodularia spp. are known to produce nodularins (NOD) and a range of other bioactive peptides such as spumigins and nodulopeptins, all of which have unclear function. In a recent study, three new nodulopeptins with molecular weight of 899, 901 and 917 were characterised from N. spumigena KAC 66. In the present study, N. spumigena KAC 66 was fractionated by reversed phase flash chromatography and their toxicity was determined by their lethality to Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. Pure NOD was lethal to D. pulex and D. magna LC50= 8.4 μg/mL and 5.0 μg/mL, respectively. The newly characterised nodulopeptin 901 was also tested against D. magna (LC50=>100 μg/mL). NOD and nodulopeptin 901 inhibited PP1 with IC50 0.038 μg/mL and 25 μg/mL, respectively. In common with many studies, the maximum amount of NOD was retained within the cells during the seven week growth experiment. In contrast, as much as ~50% of nodulopeptin 901 was detected in the growth media throughout the duration of experiments. To gain further insight on the effects of environmental stress on growth and production of bioactive metabolites in N. spumigena KAC 66, a range of parameters were investigated which included; temperature, salinity, nitrate and phosphorus. In the present study it was investigated that extreme growth conditions have a considerable effect on biomass and toxin levels by N. spumigena KAC 66. The light intensity ranged from 17.35-17.47 μmol/s/m2, 22°C and 11-20 ‰ of salinity were the optimal growth conditions to obtain maximum biomasses, intra and extracellular peptide contents. At 6.5 mg/L nitrate the maximum growth, as indicated by Chl-a and maximum concentrations of intracellular NOD and nodulopeptin 901 were detected found in week 5 and 4, respectively. Temperature had the greatest effect on peptide production. Whilst growth was similar at 22°C, 25°C and 30°C, increase in temperature had a profound effect on NOD production in that an increase from 22°C to 25°C resulted in a 50% decrease in intracellular NOD levels. At 30°C little or no NOD was detected. In contrast, whilst concentrations of nodulopeptin 901 decreased with increasing temperature, they were still detected at consistent levels suggesting they play an important role. The results from phosphate experiment showed Chl-a, cell biomass and peptide production did not show clear dependency on availability of PO-3 4. This is the first study to evaluate the effects of selected environmental parameters on NOD/nodulopeptin 901 production which ultimately may be helpful to explain the distribution, control of natural blooms and toxin levels of N. spumigena in the Baltic Sea and as well as laboratory based experiments. In an attempt further exploit cyanobacterial diversity, 20 strains were isolated from the Dian Lake and 6 from the Dead Sea. The UPLC-PDA-MS analysis of isolates, Microcystis spp. from Dian Lake, China indicated the presence of several peptides namely MC-LR, cyanopeptolin A and aerucyclamides A-D. These new isolates will be examined for biological activity and chemical characterisation in future studies.

The analysis of acute phase protein serum amyloid A (SAA) has recently been brought into clinical use in veterinary medicine. Some of the difficulties with incorporating the SAA method in clinical practice have been the expensive and rather large equipment required for the method. Due to these difficulties only larger clinics can afford to use the SAA analysis. The company Equinostic has recently developed a smaller instrument that costs one-tenth of a larger instrument. The instrument is named EVA1 and has so far only been used to analyze SAA in horses. The aim of this study was to investigate if the EVA1 instrument could be used to analyze SAA in cats. This study included 24 serum samples from cat, which were first analyzed twice on the EVA1 instrument and then sent to the Strömsholm Referral Animal Hospital in Sweden where they reanalyzed the samples using a validated reference method. Both instruments are based on an immunoturbidimetric assay. The correlation between the two instruments was good (r=0.97) but the EVA1 instrument showed constantly lower results than the reference method. The difference between the duplicates when analyzed on the EVA1 instrument was larger than expected. The conclusion is that EVA1 could be used to analyze SAA in cats. However, before it could be used clinically in veterinary practice an extended study is recommended.