No description available.

No description available.

Vascular risk factors inducing a state of chronic cerebral hypoperfusion and metabolic stress are thought to influence the onset and progression of Alzheimer’s disease (AD). To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, the first aim of this thesis was to define the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to the metabolic challenge of oxygen glucose deprivation (OGD). 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry (LC-MS). The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Ingenuity Pathway Analysis identified novel protein-protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins were associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Pertinent to AD research, amyloid binding alcohol dehydrogenase (ABAD) was found to be significantly increased in response to OGD. ABAD is emerging as a key player in mitochondrial dysfunction in AD, yet full understanding of the biochemical pathways in which this protein is involved remain elusive. Using immunoprecipitation coupled to LC-MS (IP-MS), the second aim of the thesis was to characterise the ABAD protein interactome in SH-SY5Y cells and its response to metabolic stress. 67 proteins were identified as potential ABAD interactors under control conditions, and 69 proteins were identified as potential ABAD interactors under OGD conditions. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to determine the subcellular locations and biological functions of the ABAD interacting proteins in control and OGD conditions. DAVID identified the nuclei and mitochondria to contain the greatest number of changes in ABAD interacting proteins following OGD. “Glucose Metabolic Process” (GO:0006006) was the top functional cluster for ABAD interacting proteins in both control and OGD conditions. Independent immunoprecipitations, western blotting, immunohistochemistry and electron microscopy were used to validate specific protein interactions. OGD was found to initiate a novel interaction between ABAD and glucose-regulated protein 75 (GRP75), a finding confirmed in human AD tissue. GRP75 is a mitochondrial protein and marker of the mitochondrial associated membrane (MAM), a specialised region between the mitochondria and the ER. The MAM is known to be enriched with presenilin proteins, involved in the proteolytic cleavage of amyloid precursor protein (APP). These data were used to generate an “ABAD-GRP75-MAM hypothesis of mitochondrial dysfunction in AD”, which might provide a novel link between chronic metabolic stress, ABAD, mitochondrial dysfunction and the onset / progression of AD. The third aim of the thesis was to test this novel hypothesis. Western blotting revealed APP to be significantly decreased following OGD, concurrent with an increase in ABAD protein levels. Over-expression of ABAD protein in SH-SY5Y cells was used to test whether the increased levels of ABAD following OGD were the driving force behind APP down-regulation. ABAD over-expression in SH-SY5Y cells was found to have no detectable effect on APP. Conversely, electron microscopy revealed a dynamic response of the MAM to metabolic stress. This result, along with the interaction of ABAD with GRP75, and the enrichment of presenilins at the MAM, suggests that this specialised membrane region may have an important role to play in AD.

La protéine de stress p8 fut découverte et caractérisée dans notre laboratoire. Cette protéine est surexprimée dans l'adénocarcinome pancréatique et possède un rôle dans la progression tumorale. Pendant ce travail de thèse, nous nous sommes focalisé sur le rôle de p8 dans les phases initiales de la tumorigénèse pancréatique et sur ses mécanismes d'action, impliqués dans la progression tumorale. Nous avons montré que p8 joue un rôle essentiel dans l'apparition des lésions PanINs suite à l'activation de l'oncogène Kras. Au niveau cellulaire, nous avons montré que la protéine p8 protège les cellules tumorales pancréatiques contre le stress de la privation en nutriments via la régulation de l'expression de RelB et IER3. La voie p8, RelB et IER3 est impliquée dans l'inhibition de l'apoptose suite au stress. De plus, nous avons observé que ces trois protéines sont co-exprimées dans les adénocarcinomes humains et leur expression corrèle avec l'évolution de la maladie. Dans un second lieu, nous avons démontré le rôle essentiel de la protéine p8 dans la résistance des cellules tumorales pancréatiques à l'hypoxie et à la privation en glucose. Nous avons identifié la voie p8/Aurora KinaseA, qui en réponse au stress métabolique, réduit l'apparition des dommages à l'ADN en contrôlant l'expression des gènes liés au cycle cellulaire et à la réparation de l'ADN. De plus, nos recherches ont montré que p8 protège les cellules tumorales du stress métabolique en inhibant la mort cellulaire dépendante de l'autophagie. Nous espérons que nos résultats aideront à mieux cibler les cellules tumorales pancréatiques et leur caractère résistant au stress micro-environnemental extrême. / The stress protein p8 was discovered and characterized in our laboratory. Over expressed in pancreatic adenocarcinoma, p8 is involved in tumor progression. During my PhD studies, we focused on the role of p8 in pancreatic cancer development and on its mechanisms of action. First, we demonstrated that p8 is essential for PanIN development following Kras oncogene activation. At the cellular level, we found that p8 protects pancreatic cancer cells upon nutrient starvation stress through the regulation of RelB and IER3 expression. p8, RelB and IER3-dependent cascade inhibits apoptosis after the starvation stress. Furthermore, we showed that these tree proteins are co expressed in human pancreatic adenocarcinoma. One the other hand, our study showed that p8 is involved in pancreatic cancer cells resistance to hypoxia and glucose starvation. We identified a p8/Aurora KinaseA pathway which, in response to such metabolic stress, reduces DNA damage by regulating cell cycle and DNA repair genes expression. Moreover, our studies demonstrated that p8/AURKA path protects cancer cells against metabolic stress by inhibiting autophagy-associated cell death. We expect that our data will help to get new therapeutics against pancreatic cancer.

Deficiências nutricioniais são freqüentemente associadas a aumento das taxas de morbidade e mortalidade no pós-operatório. Descreve-se há mais de 50 anos a relação existente entre perda de peso corporal pré-operatório e o aumento da mortalidade no pós-operatório. Vários fatores, além da desnutrição em si, contribuem para este quadro, entre eles a própria doença de base, a cirurgia, infecções associadas, dentre outros. Sabidamente, na presença de trauma físico e ou metabólico, são observadas alterações no metabolismo das proteínas e dos aminoácidos, além da liberação e aumento de hormônios catabolizantes. O presente trabalho objetiva avaliar se a infusão endovenosa de aminoácidos e glicose, em via periférica, em pacientes submetidos ao estresse cirúrgico, altera esse estresse e/ou resulta em retenção de nitrogênio aminoacídico. Foram estudados 18 pacientes, triados e operados pela mesma equipe médica captados do ambulatório do Serviço de Cirurgia Geral do Hospital Universitário da Universidade Federal de Alagoas. Após serem avaliados e, consentida a realização do protocolo de estudo, foram submetidos à avaliação nutricional e, aleatoriamente, divididos em dois grupos. O grupo I, ou controle, durante o período transoperatório, recebeu solução de ringer com lactato e glicose 5%, como rotineiramente utilizado pelo Serviço de Anestesia e Cirurgia da Universidade Federal de Alagoas. O grupo II, ou teste, além da infusão de soro ringer com lactato e glicose 5%, recebeu aminoácidos 6,6% e glicose 16,6%, equivalente, respectivamente, a 50 gramas de proteína e 250 g de glicose. Observou-se, nos resultados, que não houve diferença estatística entre os grupos estudados, no que se refere à sexo, faixa etária, exames bioquímicos, aminoácidos e concentração urinária de adrenalina no pré e pós-operatório. Observou-se, ainda, que o grupo II, que recebeu solução de aminoácidos e glicose, não excretou mais nitrogênio nos períodos pré, trans e pós-operatório, em comparação ao grupo I, mantendo os valores semelhantes ao grupo I após 24 horas do início da anestesia. No entanto, o grupo II, embora tenha excretado quantidades semelhantes de nitrogênio em comparação ao grupo I, teve um balanço nitrogenado, em média, menos negativo que o grupo I (p<0,05), retendo aproximadamente 40% a mais de nitrogênio aminoacídico. Conclui-se, desta forma, que a infusão parenteral de nitrogênio aminoacídico 6,6% e glicose 16,6% no transoperatório pode ser benéfico aos pacientes sob estresse cirúrgico. / It's been described for almost 50 years that weight loss at the preoperative period increases the postoperative mortality. Many factors, besides the malnutrition itself, contribute to this situation, for example, the patient illness, the surgical procedure, associated infections and others. It’s also known that in trauma, organic or not, there are disorders in proteic and aminoacid metabolism and an increase on release of catabolic hormones. This study has the objective to evaluate if the endovenous infusion of a amino acid and glucose solution, in a peripheric vein, in patients submitted to a surgical procedure, modifies the patients response to the trauma and/or the nitrogen retention. We studied 18 patients from the Surgery Division of the Clinical Hospital of the Federal University of Alagoas. The same medical team followed all patients. After evaluation and consent of the realization of the study, they went on an assessment of the nutritional status and were randomized in 2 groups. The group I (control group), during the perioperative period received a solution of lactate ringer and glucose 5%, as is usually done at this hospital. The group II (test group), besides the referred solution, received a mixture of amino acids 6,6% and glucose 16,6%, meaning 50g of protein and 250g of glucose during the transoperatory period. There was no statistic difference between the groups studied in what concerns to age, sex, biochemistry tests, aminoacids and adrenalin urinary concentration at the pre or postoperative periods. It has also been observed that group II, that received a solution with aminoacid and glucose, hasn’t excreted more nitrogen through pre, trans and postoperative periods in comparation to group I, maintaining similar values of group I 24 hours after the anesthesic procedure. However, though group II had excreted similar values of nitrogen in comparation to group I, it had a nitrogened balance less negative than group I (p<0,05) retaining approximately 40% more aminoacidic nitrogen. From that, we conclude that the parenteral infusion of aminoacidic nitrogen 6,6% and glucose 16,6% at the transoperative period may be a benefit to patients under surgical stress.

<p>Tokom perioda rane laktacije brojni unutra&scaron;nji i<br />spolja&scaron;nji faktori vr&scaron;e uticaj na produktivnost i<br />zdravlje mlečnih krava. Najznačajniji unutra&scaron;nji<br />faktori na početku laktacije su zapaljenski<br />procesi i metabolički stres. S obzirom da su<br />metaboličke i imunolo&scaron;ke promene najče&scaron;ći<br />okidač za nastanak bolesti, neophodno je bilo<br />ispitati da li postoji mogućnost regulisanja ovih<br />promena pomoću nesterodinih antiinflamatornih<br />lekova. Cilj ove doktorske disertacije je bio da<br />se ispita uticaj parenteralne primene<br />ketoprofena na metabolički status, inflamatorne<br />promene, odnos između metaboličkih i<br />inflamatornih promena kod krava posle teljenja,<br />kao i na proizvodnju mleka kod krava u ranoj<br />laktaciji. Za ogled je odabrano 30 krava.<br />Oglednoj grupi od 15 krava je i.m. aplikovan<br />ketoprofen u terapijskoj dozi (3mg/kg telesne<br />mase) tokom tri uzastopna dana nakon teljenja.<br />Kontrolna grupa od 15 krava je predstavljala<br />negativnu kontrolu. Uzorci krvi za analizu su<br />uzimani iz repne vene (vena coccygea) na dan<br />teljenja, u prvoj i drugoj nedelji posle teljenja.<br />Procena inflamacije je vr&scaron;ena na osnovu<br />određivanja koncentracije proteina akutne faze<br />(haptoglobina i fibrinogena) i proinflamatonih<br />citokina (interleukina-1&alpha;, faktora nekroze<br />tumora-alfa i interferona gama), dok je procenametaboličkog statusa vr&scaron;ena na osnovu određivanja vrednosti metaboličkih parametarau svim uzorcima krvnog seruma i u svim nedeljama ogleda. Ketoprofen primenjivan parenteralno kod krava posle teljenja je ublažio metabolički stres, uticao na smanjenje inflamatornih procesa i na smanjnje međusobne povezanosti i uticaja između metaboličkih i inflamatornih promena kod krava posle teljenja u poređenju sa netretiranim kravama, a imao je i pozitivan uticaj na proizvodnju mleka u ranoj laktaciji.</p> / <p>During the period of early lactation numerous<br />internal and external factors influence the health<br />and productivity of dairy cows. The most<br />important internal factors at the beginning of<br />lactation are inflammatory processes and<br />metabolic stress. Since the metabolic and<br />immunologic changes are the most common<br />trigger for the disease, it was necessary to<br />examine whether there is a possibility of<br />regulating these changes by using non-steroidal<br />anti-inflammatory drugs. The aim of this<br />doctoral thesis was to investigate the effect of<br />parenteral administration of ketoprofen in the<br />metabolic status, inflammatory changes, the<br />relationship between metabolic and<br />inflammatory changes in cows after calving and<br />milk production in cows in early lactation. This<br />study included 30 cows. Experimental group of<br />15 cows is treated i.m. ketoprofen administered<br />in a therapeutic dose (3 mg / kg body weight)<br />for three consecutive days after calving. A<br />control group of 15 cows accounted for a<br />negative control. Blood samples for analysis<br />were taken from the tail vein (vena coccygea)<br />on the day of calving, in the first and second<br />week after calving. Evaluation of inflammation<br />was based on the determination of the<br />concentration of acute phase proteins<br />(haptoglobin and fibrinogen) and<br />proinflamatonih cytokines (interleukin-1&alpha;,tumor necrosis factor-alpha and interferongamma),and the evaluation of the metabolic status performed on the basis of determining the value of metabolic parameters in a blood sample serum and in all the weeks of the experiment. Ketoprofen administered parenterally in cows after calving eased metabolic stress, had impact on reducing inflammatory processes and the decrease of interconnection and influence between metabolic and inflammatory changes in cows after calving compared with untreated cows, and had a positive impact on milk production in the early lactation.</p><p>&nbsp;</p>

The central complex is a prominent neuropil found in the middle of the insect brain. It is considered as a higher center for motor control and information processing. Multiple neuropeptides and neurotransmitters are produced in neurons of the central complex, however, distribution patterns and functional roles of signaling substances in this brain region are poorly known. Thus, this thesis focuses on the distribution of signaling substances and on modulatory roles of neuropeptides in the central complex of Drosophila. Immunocytochemistry in combination with GAL4/UAS technique was used to visualize various signaling substances in the central complex. We revealed different central-complex neurons expressing the neuropeptides; Drosophila tachykinin (DTK), short neuropeptide F (sNPF), myoinhibitory peptide (MIP), allatostatin A, proctolin, SIFamide, neuropeptide F and FMRFamide. Subpopulations of DTK, sNPF and MIP-expressing neurons were found to co-localize a marker for acetylcholine. In addition, five metabotropic neurotransmitter receptors were found to be expressed in distinct patterns. Comparison of receptor/ligand distributions revealed a close match in most of the structures studied. By using a video-tracking assay, peptidergic modulation of locomotor behavior was studied. Different DTK and sNPF-expressing neurons innervating the central complex were revealed to modulate spatial distribution, number of activity-rest phases and activity levels, suggesting circuit dependent modulation. Furthermore, neurosecretory cells in the Drosophila brain that co-express three types of neuropeptides were shown to modulate stress responses to desiccation and starvation. In summary, we have studied two different neuropeptides (DTK and sNPF) expressed in interneuronal circuits and neurosecretory cells of the Drosophila brain in more detail. We found that these neuropeptides display multiple actions as neuromodulators and circulating hormones, and that their actions depend on where they are released. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript.

Software sensors are a potent tool to improve biotechnological real time process monitoring and control. In the current project, algorithms for six partly novel, software sensors were established and tested in a microbial reactor system. Eight batch and two fed-batch runs were carried out with a recombinant Escherichia coli to investigate the suitability of the different software sensor models in diverse cultivation stages. Special respect was given to effects on the sensors after recombinant protein expression was initiated by addition of an inducer molecule. It was an objective to figure out influences of excessive recombinant protein expression on the software sensor signals. Two of the developed algorithms calculated the biomass on-line and estimated furthermore, the specific growth rate by integration of the biomass changes with the time. The principle of the first was the application of a near infrared probe to obtain on-line readings of the optical density. The other algorithm was founded on the titration of ammonia as only available nitrogen source. The other two sensors analyzed for the specific consumption of glucose and the specific production of acetate and are predicted on an in-line HPLC system. The results showed that all software sensors worked as expected and are rather powerful to estimate important state parameters in real time. In some stages, restrictions may occur due to different limitation affects in the models or the physiology of the culture. However, the results were very convincing and suggested the development of further and more advanced software sensor models in the future.

Force production by a muscle is critical to maintaining proper function and overall health of a human or animal. Muscle adapts to increased loading with hypertrophy by activating a number of intracellular signaling cascades that regulate protein synthesis. The overall hypothesis is that force-dependent processes acutely activate growth-related signaling during active force generation. This project took two approaches. The first employed a general survey of muscles in which age-dependent changes in muscle activity differed. No conclusive activity-dependent signaling emerged however coordinated signaling among kinases broke down with age. The second approach utilized an in situ muscle preparation in which force production or metabolic costs were specifically controlled. Similar sub-maximal force levels generated by different methods found that force, per se, is not a primary modulator of growth-related signaling but that ERK phosphorylation is dependent on fiber-activation. Prolonging the duration of electrical stimulation applied to the nerve or increasing the frequency at which stimulations are applied was expected to increase the metabolic stress associated with contraction. Several growth-related kinases correlated with markers of metabolic stress, i.e. increased AMPK activity and decreased glycogen content, which were decoupled from force decline. This suggests energy depletion, specific to stimulation pattern, strongly influences the immediate response to high force contraction signaling. The overall conclusion is that signaling molecules previously implicated in force-dependent signaling lie much too downstream to relay strict force-dependent signaling.

<p>Software sensors are a potent tool to improve biotechnological real time process monitoring and control. In the current project, algorithms for six partly novel, software sensors were established and tested in a microbial reactor system. Eight batch and two fed-batch runs were carried out with a recombinant <em>Escherichia coli</em> to investigate the suitability of the different software sensor models in diverse cultivation stages. Special respect was given to effects on the sensors after recombinant protein expression was initiated by addition of an inducer molecule. It was an objective to figure out influences of excessive recombinant protein expression on the software sensor signals.</p><p>Two of the developed algorithms calculated the biomass on-line and estimated furthermore, the specific growth rate by integration of the biomass changes with the time. The principle of the first was the application of a near infrared probe to obtain on-line readings of the optical density. The other algorithm was founded on the titration of ammonia as only available nitrogen source. The other two sensors analyzed for the specific consumption of glucose and the specific production of acetate and are predicted on an in-line HPLC system.</p><p>The results showed that all software sensors worked as expected and are rather powerful to estimate important state parameters in real time. In some stages, restrictions may occur due to different limitation affects in the models or the physiology of the culture. However, the results were very convincing and suggested the development of further and more advanced software sensor models in the future.</p>