Дисертації з теми "MRNA fate"

TDP43 is an ubiquitously expressed RNA-binding protein implicated in several aspects of RNA metabolism. It can shuttle between the nucleus and the cytoplasm; however, when it is mutated in some familial Amyotrophic Lateral Sclerosis (ALS) cases, it undergoes nuclear clearance and cytoplasmic accumulation, driving neuronal degeneration. The same phenotype is present in patients bearing ALS-inducing mutations in other genes and ALS sporadic patients, defining TDP43 proteinopathy as a common feature in this pathology. Why does it cause specific motor neuron death? Our quantitative proteomics analysis of the TDP43 interactome revealed the interaction with components of the mRNA surveillance pathway, suggesting a still undiscovered function in nonsense-mediated decay. We demonstrated that TDP43 acts translation- and SMG1-dependently as a mRNA decay enhancer of specific transcripts by binding their 3’UTR. In particular, it leads to the down-regulation of transcripts with a long 3’UTR. From our sequencing data of spinal cords from TDP43Q331K transgenic mouse model and of motor neuron-like NSC-34 cells silenced for TDP43 emerged that TDP43 plays another striking role in the 3’UTR, modulating mRNA alternative polyadenylation and promoting the generation of shorter transcripts. This finding is supported by the direct interaction of TDP43 with the cleavage stimulation factor, a core component of the polyadenylation machinery. These results broaden our knowledge of the role of TDP43 in the post-transcriptional gene expression regulation. The impairment of these two biological processes by TDP43 proteinopathy could have implications in ALS pathogenesis, representing possible new targets for therapeutic approaches.

TDP43 is an ubiquitously expressed RNA-binding protein implicated in several aspects of RNA metabolism. It can shuttle between the nucleus and the cytoplasm; however, when it is mutated in some familial Amyotrophic Lateral Sclerosis (ALS) cases, it undergoes nuclear clearance and cytoplasmic accumulation, driving neuronal degeneration. The same phenotype is present in patients bearing ALS-inducing mutations in other genes and ALS sporadic patients, defining TDP43 proteinopathy as a common feature in this pathology. Why does it cause specific motor neuron death? Our quantitative proteomics analysis of the TDP43 interactome revealed the interaction with components of the mRNA surveillance pathway, suggesting a still undiscovered function in nonsense-mediated decay. We demonstrated that TDP43 acts translation- and SMG1-dependently as a mRNA decay enhancer of specific transcripts by binding their 3’UTR. In particular, it leads to the down-regulation of transcripts with a long 3’UTR. From our sequencing data of spinal cords from TDP43Q331K transgenic mouse model and of motor neuron-like NSC-34 cells silenced for TDP43 emerged that TDP43 plays another striking role in the 3’UTR, modulating mRNA alternative polyadenylation and promoting the generation of shorter transcripts. This finding is supported by the direct interaction of TDP43 with the cleavage stimulation factor, a core component of the polyadenylation machinery. These results broaden our knowledge of the role of TDP43 in the post-transcriptional gene expression regulation. The impairment of these two biological processes by TDP43 proteinopathy could have implications in ALS pathogenesis, representing possible new targets for therapeutic approaches.

Cardiovascular disease (CVD) is still one of the major cause of morbidity and mortality in the world, with ischemic heart disease representing the majority of deaths over the past 10 years. The high burden of the disease, both immediate and chronic, associated with the high costs for the healthcare systems, claim for the development of novel therapeutic strategies. The main issue of current pharmacological and interventional therapeutic approaches is their inability to compensate the great and irreversible loss of functional cardiomycytes (CMs). Because of the limited regenerative capacity of post-natal CMs and the difficulty to obtain and isolate heart bioptic tissue, very limited supplies of these cells are available at present for dedicated studies. Moreover, even if animal models are surely the best tool to study and understand in vivo the mechanisms of specific human pathologies in a complex organism, they are not fully predictive and representative of the human condition; from an economic point of view, animal maintenance and the related experimentations are time consuming and very expensive. In this scenario, human pluripotent stem cells (hPSCs), including human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs), play an important role in the cardiovascular research field, because they can be indefinitely expanded in culture without loosing their stemness, and differentiated into cells of the three germ layers, such as CMs. A great breakthrough in science has occurred in 2007 with the discovery of hiPSCs by the Nobel Prize Shinya Yamanaka. This has been the starting point for deriving patient-specific hiPSCs from the reprogramming of somatic cells obtained with less- or non-invasive procedures (skin biopsies, blood, urine…), useful for the generation of tissues for autologous-repair, bypassing the ethical and political debates surrounding the hESCs derivation. The researchers have made several efforts to develop strategies to efficiently direct hPSCs cardiac differentiation and the existing methods for deriving CMs involve stage-specific perturbations of different signaling pathways using growth factors (GFs) or small molecules that recapitulate key steps of the cardiac development observed in vivo. However, these strategies are accompanied by some limitations including: high intra- and inter-experimental variability, low efficiencies, presence of xeno-contaminants, undefined medium components and differences in the expression of cytokines of endogenous signaling pathways. Other strategies are based on the direct lineage conversion of somatic cells, especially fibroblasts, via the overexpression of cardiac transcription factors (TFs) combinations through integrating and non-integrating vectors. However, also these approaches are characterized by low efficiencies, combined with the risk of genomic integration and insertional mutagenesis when using integrating vectors or the need for stringent steps of purification when using non-integrating techniques. Because of the difficulty to specifically direct hPSCs cardiac fate in a robust way, combined with the scarce ability of conventional culture systems to reproduce in vitro, the environment in which cells reside in vivo, the CMs produced to date are immature and more similar to fetal cardiac cells. In 2010, Warren L. and co-workers pioneered a novel, non-integrating strategy based on repeated transfection with cathionic vehicles of synthetic modified messenger RNA (mmRNA), specifically designed to avoid innate immune response from the cell, demonstrating the possibility to both reprogram somatic cells to pluripotency and to programm hPSCs fate into terminally differentiated myogenic cells. Hence, the aim of this PhD thesis is the development of an efficient and robust method for cardiac differentiation of hPSCs by combining the mmRNA with the microfluidic technology. Repeated transfections with mmRNA encoding 6 cardiac TFs are employed to force the endogenous protein expression in the cells and to drive the differentiation toward functional maturation of CMs. The integration of cardiac differentiation within an ad hoc microfluidic platform, facbricated in BioERA laboratory, allows a more precise control of culture conditions, enabling a high mmRNA transfection efficiency, thanks to the high volume/surface ratio, and the in vitro reproduction of physiological niches. In fact, the small scale offered by microfluidics, best mimics the cellular dynamics, which occur in the soluble microenvironment in vivo. Moreover, the microfluidic technology offers the possibility to perform combinatorial, multiparametric, parallelized and highthroughput experiments at one time in a cost-effective manner, not achievable and not economically sustainable in macroscopic conventional culture systems. Chapter 1 starts with the definition of regenerative medicine and introduces the complexity of cardiac development, with the network of TFs that cooperate in this process. The state of the art regarding the derivation of CMs from hPSCs and from the transdifferentiation of somatic cells is described, together with the current limitations and challenges. Finally, the general aim of this PhD thesis is presented. Chapter 2 will focus on hPSCs (hES and hiPS) employed during this project, describing their most important characteristics. It will be also presented a monolayer-based cardiac differentiation protocol of hPSCs that, to date is considered the gold standard for the fast generation of a high yield of beating CMs in conventional culture systems. This protocol relies on the temporal modulation of Wnt pathway via the administration of small molecules. In addition, a hES line, dual reporter for 2 cardiac TFs will be described and always adopted as a tool to monitor the progression of cardiac differentiation. The results obtained in standard cultures will be showed. Chapter 3 will review the state of the art of microfluidic technology for cell culture in regenerative medicine applications. Then, the microfluidic platform fabrication will be described and employed, followed by the optimization of culture, expansion and cardiac differentiation of hPSCs with the gold standard protocol deriving form the translation from macro- to micro-scale. Chapter 4 will introduce the novel mmRNA strategy for reprogramming and programming cell fate: also in this case the state of the art will be discussed. Then, the experimental strategies developed to program cardiac differentiation of hPSCs toward a more mature CM phenotype will be presented, together with the results obtained and the related structural, functional and molecular characterizations. In this work, for the first time, it has been possible to derive CMs from hPSCs with repeated transfections of mmRNA encoding 6 cardiac TFs in microfluidics, with efficiencies higher to current methods described in literature, performed in standard systems. Finally, Chapter 5 will present the general discussion and conclusions, with the future perspectives regarding the use of mmRNA combined with microfluidic technology for deriving different CMs phenotypes, just varying the combination of TFs delivered. To conclude, the experiments developed during this project provide proof-of-principle that it is possible to program hPSCs fate toward cardiac lineage and cardiac maturation in microfluidics; moreover, thanks to the non-integrating characteristic of mmRNA, the CMs obtained are clinical-grade and could potentially be employed in the next future for clinical applications of autologous tissue self-repair and for personalized drug screening.
Le malattie cardiovascolari rappresentano ad oggi una delle principali cause di morbidità e mortalità nel mondo, tra le quali la patologia ischemica è responsable del maggior numero di decessi negli ultimi 10 anni. L’elevato impatto determinato da tali patologie, sia acute che croniche, e gli elevati costi per i sistemi sanitari, richiedono lo sviluppo di nuove strategie terapeutiche. La questione principale riguardante gli attuali approcci terapeutici, sia farmacologici sia interventistici, è rappresentata dalla loro incapacità di compensare l’elevata ed irreversibile perdita di cardiomiociti funzionali. A causa della limitata capacità rigenerativa dei cardiomiociti post-natali e della difficoltà di reperire ed isolare tessuto cardiaco bioptico, scarse sono le fonti di tali cellule disponibili per uno studio dedicato. Tra l’altro, anche se i modelli animali ancora oggi rappresentano sicuramente lo stumento migliore per studiare e comprendere in vivo i meccanismi alla base dello sviluppo di specifiche patologie umane, nel constesto di un organismo complesso, essi non sono completamente predittivi e rappresentativi della condizione umana analizzata; da un punto di vista economico, il mantenimento di tali animali e le relative sperimentazioni, richiedono molto tempo e costi elevati. In questo scenario, le cellule staminali umane pluripotenti (hPSCs), comprese le cellule staminali embrionali (hESCs) e le cellule staminali pluripotenti indotte (hiPSCs), rivestono un ruolo importante nella ricerca cardiovascolare perché possono essere espanse in coltura indefinitamente, senza perdere la loro staminalità, e differenziare nelle cellule che componogono i tre foglietti germinativi, come ad esempio i cardiomiociti. Un’importante svolta nella ricerca scientifica è avvenuta nel 2007, con la scoperta delle hiPSCs da parte del Premio Nobel Shinya Yamanaka. Ciò ha rappresentato il punto di partenza per derivare hiPSCs paziente-specifiche attraverso il reprogramming di cellule somatiche ottenute con procedure mini- o non-invasive (derivate da biopsie cutanee, sangue, urina…), utili per generare tessuti per una riparazione autologa, evitando i problemi etici e politici relativi alla derivazione delle hESCs. Notevoli studi sono stati condotti dai ricercatori nel tentativo di sviluppare strategie che efficientemente ed in maniera robusta guidino il differenziamento cardiaco delle hPSCs, basate sulla perturbazione stadio-specifica di differenti vie di segnalazione, mediante l’uso di fattori di crescita e piccole molecole, che ricapitolano i punti essenziali dello sviluppo cardiaco osservato in vivo. Tuttavia, questi metodi sono accompagnati da alcune limitazioni, quali: elevata variabilità intra ed inter-sperimentale, presenza di xeno-contaminanti, componenti indefinite nei medium di coltura e differenze nei livelli di espressione di citochine endogene. Altre strategie si basano invece sulla conversione diretta di cellule somatiche, specialmente fibroblasti, attraverso l’overespressione di una combinazione di fattori di trascrizione cardiaci mediante vettori integrativi e non-integrativi; tuttavia, anche tali approcci sono caratterizzati da basse efficienze nella generazione di cardiomiociti, associate al rischio di integrazioni genomiche e mutagenesi inserzionale nel caso dei vettori integrativi, o alla necessità di effettuare diversi step di purificazione quando si ultilizzano sistemi non-integrativi. Pertanto, a causa delle difficoltà dei sistemi convenzionali di coltura nel dirigere specificamente ed in maniera robusta il differenziamento cardiaco delle hPSCs, assieme alla scarsa capacità di riprodurre in vitro l’ambiente in cui le cellule risiedono in vivo, i cardiomiociti prodotti attualmente sono immaturi e più simili allo stadio fetale di sviluppo. Nel 2010 Warren L. ed il suo gruppo di ricerca ha sperimentato per la prima volta una tecnologia innovativa di tipo non-integrativo basata su trasfezioni ripetute con lipidi cationici di RNA messaggeri modificati sinteticamente (mmRNA) per evitare la risposta immunitaria innata da parte delle cellule; egli ha dimostrato la possibilità sia di riprogrammare cellule somatiche allo stato pluripotente, sia di programmare il differenziamento miogenico di hiPSCs. Pertanto, lo scopo di questa tesi di dottorato è quello di sviluppare un metodo robusto ed efficiente per il differenziamento cardiaco di hPSCs combinando gli mmRNA con la tecnologia microfluidica. Ripetute trasfezioni di mmRNA codificanti per 6 fattori di trascrizione coinvolti nello sviluppo e nel funzionamento cardiaco, vengono impiegate per forzare l’espressione proteica endogena delle cellule e per guidare il differenziamento verso la maturazione funzionale dei cardiomiociti. L’integrazione del differenziamento cardiaco in una piattaforma microfluidica ad hoc, prodotta nel laboratorio BioERA, consente un controllo più preciso delle condizioni di coltura garantendo un’elevata efficienza di trasfezione degli mmRNA grazie all’elevato rapporto superficie/volume e permette la riproduzione in vitro di nicchie fisiologiche. Infatti, la miniaturizzazione consente di mimare al meglio le dinamiche cellulari che avvengono in vivo nel microambiente solubile. Le tecnologia microfluidica offre la possibilità di effettuare esperimenti combinati, multiparametrici e paralleli in una sola volta e con elevato rendimento a costi ridotti, non realizzabili nei macroscopici e costosi sistemi di coltura convenzionali. Il Capitolo 1 inizia con la definizione di medicina rigenerativa e introduce la complessità dello sviluppo cardiaco ed il network di fattori di trascrizione che cooperano durante questo processo. Viene poi descritto lo stato dell’arte relativo alle strategie per l’ottenimento di cardiomiociti da hPSCs e al transdifferenziamento cardiaco di cellule somatiche, insieme alle relative limitazioni e alle problematiche attuali da risolvere. Infine viene presentato lo scopo generale di questa tesi di dottorato. Il Capitolo 2 si focalizzerà sulle hPSCs (sia hES sia hiPS) impiegate durante questo progetto, descrivendo le caratterisatiche principali di tali cellule. Verrà inoltre presentato un protocollo di differenziamento cardiaco di hPSCs in monostrato che attualmente è considerato il gold standard per ottenere velocemente un’elevata resa di cardiomiociti contrattili in supporti di coltura convenzionali. Tale protocollo si basa sulla modulazione del pathway canonico di Wnt attraverso l’applicazione di due piccole molecole. Inoltre, una linea di hES, doppio reporter per 2 fattori di trascrizione cardiaci, verrà descritta ed impiegata in tutti gli esperimenti come strumento per monitorare l’andamento del differenziamento cardiaco delle hPSC. I risultati ottenuti in colture standard verranno mostrati. Il Capitolo 3 esaminerà lo stato dell’arte della tecnologia microfluidica nelle applicazioni di medicina rigenerativa, sottolineando i vantaggi derivanti dalla combinazione della microtecnologia con la biologia cellulare. Verrà successivamente descritta la fabbricazione della piattaforma microfluidica utilizzata, con la successiva ottimizzazione della coltura, espansione e differenziamento cardiaco gold standard delle hPSCs conseguenti alla conversione dalla macro- alla microscala. Il Capitolo 4 introdurrà la nuova strategia degli mmRNA per la riprogrammazione e la programmazione cellulare: anche in tal caso verrà discusso lo stato dell’arte. In seguito, verranno presentate le strategie sperimentali sviluppate per programmare il differenziamento cardiaco delle hPSCs verso un fenotipo più maturo dei cardiomiociti, insieme ai risultati ottenuti con le relative caratterizzazioni strutturali, funzionali e molecolari. In questo lavoro, per la prima volta, è stato possibile ottenere cardiomiociti da hPSCs attraverso ripetute trasfezioni di mmRNA per 6 fattori di trascrizione cardiaci in microfluidica, con efficienze superiori rispetto ai metodi presenti attualmente in letteratura, svolti in sistemi convenzionali. Il Capitolo 5 infine presenterà la discussione e le conclusioni generali, assieme alle prospettive future riguardanti l’uso degli mmRNA combinati con la microfluidica per ottenere diversi fenotipi di cardiomiociti, variando la combinazione di fattori di trascrizione veicolati. In conclusione, gli esperimenti sviluppati in questo progetto di dottorato forniscono un proof-of-principle della possibilità di programmare con gli mmRNA il destino delle hPSCs verso il differenziamento e la maturazione di cardiomiociti funzionali in microfluidica; inoltre, essendo gli mmRNA una strategia non-integrativa , i cardiomiociti ottenuti in questo modo possono essere impiegati nel prossimo futuro per applicazioni cliniche di ricostruzione tissutale autologa e per screening farmacologici personalizzati.

Sirtuins (SIRT) regulate the transcription of various genes involved in the development of diet-induced obesity and chronic disease; however, it is unknown how they change acutely following a high-fat meal. The purpose of this study was to determine the effect of a high-fat meal (65% kcals/d; 85% fat recommendation), on SIRT1-7 mRNA expression in blood leukocytes at 1, 3, and 5-h post-prandial. Men and women (N=24) reported to the lab following an overnight fast (>12H). Total RNA was isolated and reverse transcribed prior to using a Taqman qPCR technique with 18S rRNA as a normalizer to determine SIRT1-7 mRNA expression. An additional aliquot of serum was used to measure triglycerides. Data was analyzed using a RM ANOVA with P<0.05. Triglycerides (P<0.001; 124%) peaked at 3-h. SIRT 1 (P=0.004; 70%), and SIRT 6 (P=0.017; 53%) decreased expression at 3-h. SIRT4 (P=0.024) peaked at 5H relative to baseline (70%) and 3-h (68%). To our knowledge, this is the first study to report that consumption of a high-fat meal transiently alters SIRT mRNA expression consistent in a pattern that mirrors changes in serum triglycerides. Decrease in expression of SIRT1 and SIRT6 combined with an increased SIRT4 would be consistent with an increase in metabolic disease risk if maintained on a chronic basis.

Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies. This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur. Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone. Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer. Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs.
Ph. D.

Problemstellung: Die Tiergesundheit hat sich bei Milchkühen in den letzten Jahren weltweit negativ entwickelt. Wichtigster Ausdruck dafür ist die auf ca. 2,4 Jahre verkürzte Nutzungsdauer. Dabei spielt das Fettmobilisationssyndrom eine dominante Rolle. Das Fettgewebe ist nicht nur als reiner Energiespeicher, sondern als endokrines stoffwechselaktives Organ anzusehen. Untersuchungen an Menschen und Mäusen haben gezeigt, dass das Fettgewebe in Abhängigkeit von seiner Lokalisation im Körper unterschiedlich auf metabolische und hormonelle Stimuli reagiert. Es gibt Hinweise, dass auch für das Rind ähnliche Differenzen angenommen werden können. Zielstellung: Um die Eigenschaften des bovinen Fettgewebes und seine Rolle im Energiestoffwechsel besser charakterisieren zu können, war das Ziel der vorliegenden Untersuchung, die mRNA-Expressionen ausgewählter für den Fettstoffwechsel relevante Gene im bovinen Fettgewebe an verschiedenen Lokalisationen grundlegend in gesunden Rindern zu untersuchen. Material und Methoden: Die Probenentnahme erfolgte an 12 gesunden Schlachtkühen direkt nach der Tötung, die aufgrund Schwermelkbarkeit oder Unfruchtbarkeit geschlachtet wurden. Das Fettgewebe wurde aus dem Omentum majus, dem Depotfett der Niere, im kaudalen Beckendrittel (retroperitoneales Fett), dem Hüftbereich (subkutanes Fett) und dem Fett an der Herzbasis entnommen. Die Proben wurden in Flüssigstickstoff tiefgefroren, auf Trockeneis transportiert und bis zur Untersuchung bei -70°C gelagert. Die mRNA-Expression für die verschiedenen Gene (Hormonsensitive Lipase (HSL), Lipoproteinlipase (LPL), Fettsäuresynthase (FASN), Leptin, Adiponektin, Retinolbindungsprotein 4 (RBP4), Tumornekrosefaktor  (TNF) und Interleukin 6 (IL-6), Fettsäurebindungsproteine (FABP3, 4 und 5) und Glukosetransporter 4 (GLUT4)) , wurden mit einer quantitativen real time (RT)-PCR gemessen. Ergebnisse: Die mRNA-Expressionen der verschiedenen oben genannten Gene, ausgenommen IL-6 und FABP3, sind im bovinen Fettgewebe nachweisbar. Die mRNA-Expressionen unterschieden sich in den einzelnen Fettdepots nicht signifikant. Ausnahme hierbei bildete RBP4, dessen mRNA im pericardialen Fett signifikant höher exprimiert war als im subkutanen und omentalen Depot. Die mRNA-Expression des subkutanen, omentalen, perirenalen und pericardialen Fettdepots korrelierten signifikant positive untereinander. Schlussfolgerung: Die mRNA-Expressionen der in den Fettstoffwechsel involvierten und untersuchten Gene gesunder Rinder waren nachweisbar, unterschieden sich jedoch nicht signifikant von einander mit Ausnahme der RBP4 mRNA. Die positiven signifikanten Korrelationen zwischen dem subkutanen, omentalen, perirenalen und pericardialen Fettlokalisationen und gleichmäßigen Expressionen innerhalb der Gewebe deuten auf eine einheitliche Fettmetabolismus des gesamten Körpers. Verglichen mit Ergebnissen der Humanmedizin sind nur wenige Übereinstimmungen (HSL, LPL, GLUT4,TNF) zu eruieren. Weitere Studien mit gesunden Tieren im Vergleich zu erkrankten Rindern müssen klären, ob eine mögliche Verschiebung der mRNA-Konzentrationen auf das Fettmobilisationssyndrom hinweisen
Purpose: Over the last years, the situation of animal health concerning dairy cows has developed worldwide in an adverse way. Most important indicator is the shortened useful life of approx. 2.4 years. The fat mobilization syndrome plays a dominant role in this process. Apparently, fatty tissue does not only serve as a mere energy reservoir, but also as an endocrin organ with metabolic activity. Researches on humans and mice have shown fatty tissue to react on metabolic and hormonal stimuli in different ways, depending on its body localization. There are dues to anticipate, similar differences in cattle. Objectives: In order to better characterize the attributes of bovine fatty tissue and its purpose in metabolism, the present study aims examine basically the expression of mRNA in selected genes which are important for lipid metabolism in bovine fatty tissue of different localizations in healthy cattle. Methods and material: Samples where taken from twelve carcasses of healthy dairy cows slaughtered for reason of difficult milking or infertility directly after killing. Fatty tissue was taken from omentum major, kidney capsula, caudal pelvis area (retroperiteonal fat), hip area (subcutaneous fat), and cardiac base. It was instantly quick-freezed in liquid nitrogen, put on dry ice while transporting, and stored at -70°C until analysis. The expression of mRNA of different genes (hormone-sensitive lipase (HSL), lipoproteine lipase (LPL), fatty acid synthase (FASN), fatty acid binding proteine (FABP3,4 and 5), retinol binding proteine 4 (RBP4), adiponectine, glucose transporter 4 (GLUT4), leptin, interleukin-6 (IL-6), and tumor necrosis factor a (TNFα) was measured by means of a quantitative real-time (RT)-PCR. Results: The mRNA-expressions of all these different genes except IL-6 and FABP3 were detected in bovine fatty tissue. The differences of mRNA-expression between sample localization were not statistically significant. RBP4 was excepted, which mRNA showed a significantly higher expression in pericardial fat than in subcutaneous and omental fat, respectively. The correlation between mRNA-expressions of subcutaneous, omental, pericardial and perirenal fat was significant. Conclusions: The mRNA-expression of examined genes being involved in fatty tissue metabolism, were detected in healthy cattle, but were not significantly different, except RBP4. Significantly positive correlations between subcutaneous, omental, perirenal and pericardial localization and consistent expression indicate an integrative metabolism of the whole body. Compared to results of the human medicine only few analogies (HSL, LPL, GLUT4, TNF) were found. Further studies comparing healthy and diseased cattle will have to prove, if possible displacements of the mRNA-level can indicate the fat mobilization syndrome being present

The effect of exogenous unsaturated fatty acids on cellular fatty acid biosynthesis in mammary cells was examined. Under normal situations, even though the diet of a dairy cow contains considerable amounts of unsaturated fatty acids, viz. oleic acid (18:1) and linoleic acid (18:2), the major 18-carbon fatty acid that enters the circulation post-ruminally for delivery to the mammary gland is saturated fatty acid, viz. stearic acid (18:0). This is due to extensive ruminal biohydrogenation of unsaturated fatty acids. Studies have indicated that saturated fatty acids such as 18:0 are enhancers and that certain unsaturated fatty acids are inhibitors of de novo fatty acid synthesis in tissues such as the liver and adipose tissue. The present study investigated the effect of cis and trans isomers of 18:1 and 18:2 on de novo fatty acid synthesis and desaturation in mouse and bovine mammary epithelial cell cultures, and compared it with the effect caused by 18:0. In the first experiment 12.5, 25, 50 or 100 micromoles stearic acid (SA), oleic acid (OA), elaidic acid (EA), trans-vaccenic acid (TVA), linoleic acid (LA) or conjugated linoleic acid (CLA) were supplemented in the media of mouse mammary epithelial (MME) cells that were grown to confluence in Dulbecco's modified Eagle's medium (DMEM). As indicated by cellular palmitic acid (16:0) content and fatty acid synthetase (FAS) activity, when compared with SA all unsaturated fatty acid treatments inhibited de novo fatty acid synthesis in MME cells. In addition, OA at all concentrations and LA and CLA at 50 and 100 micromoles inhibited cellular stearoyl-CoA desaturase (SCD) activity and mRNA abundance. However, EA and TVA, when compared with SA, enhanced SCD activity and mRNA abundance at 12.5 and 25 micromoles. In the second experiment 25, 50 or 100 micromoles SA, OA, TVA, LA or CLA were supplemented in the media of bovine mammary epithelial cells that were grown to confluence in DMEM. As indicated by cellular 16:0 content, acetyl-CoA carboxylase (ACC) activity and FAS activity, treatment with the unsaturated fatty acids inhibited de novo fatty acid synthesis at all concentrations, when compared with SA. Unsaturated fatty acid treatments also reduced the abundance of ACC and FAS mRNA in the cells. When compared with SA at all treatment-concentrations, OA and LA inhibited whereas TVA and CLA enhanced cellular SCD activity and mRNA abundance in the bovine cells. In both cell types, CLA and TVA appeared to be the most potent inhibitors of saturated fatty acid biosynthesis.
Ph. D.

Methicillin resistant Staphylococcus aureus (MRSA) has become one of the major public health challenges worldwide. MRSA strains are capable of causing from mild non-life-threatening to severe infections of the skin and soft tissues, and even death. Skin infections caused by MRSA include primary pyodermas such as folliculitis, furuncles, carbuncles, and impetigo. Infections involving the soft tissues include cellulitis and pyo-myositis, which are less common but can cause serious morbidity. The contamination of raw food, especially meat and milk, by MRSA is well documented as well as its transmission to humans via animal contact. In addition, humans can act as reservoirs of MRSA without showing any clinical signs, thus they can contaminate foods by handling. Its presence in several kinds of food has suggested the possibility of MRSA to act as a foodborne pathogen. Although the hypothesis is suggestive, there are insufficient evidence to consider the foodstuff a vehicle of MRSA infection. For instance, nothing is known about its ability to survive under human gastroenteric conditions. Despite of the potential hazard for human health there is a lack of date on prevalence of MRSA in some foods, such as buffalo milk and buffalo dairy products. The high consumptions of buffalo drinking milk and dairy products worldwide involve an elevated number of consumers of all ages, and this is crucial considering their potential role in the transmission of foodborne pathogens. To address these issues, the aims of the thesis are: i) to assess the occurrence of MRSA in new ecological niches such as buffalo dairy farms and buffalo tank milk from Italy in order to better understand the epidemiology of MRSA ii) to study the fate of MRSA strains isolated from foods and from humans along the human gastrointestinal tract and its inter-species interaction with the human gut microbiota. Regarding the occurrence of MRSA in raw buffalo milk, seventy-five bulk tank milk (BTM) samples from farms and 24 nasal swabs from farm workers were collected, respectively. Three (4%) out of 75 BTM samples and 1 (4%) out of 24 nasal swabs were MRSA-positive. The milk isolates showed the following genotypes: ST1/t127/Va and ST72/t3092/V, while the human isolate was characterized as ST1/t127/IVa. No ST398 were found. All the isolates were multidrug resistant but vancomycin susceptible, carrying the icaA gene, while they tested negative for pvl and ses genes. This study demonstrates for the first time in Europe that MRSA might be present in dairy buffalo farms and in raw buffalo milk. For what concern the second aim, a MRSA ST398/t011/V strain, previously isolated from raw cow milk, and a human origin MRSA strain were inoculated into two foods of animal origin respectively. The pH of the matrices was gradually decreased to 2.0 in 2 hours, during which time they were kept at 37°C and periodically homogenized. The same MRSA strains levels were inoculated within an intestinal in vitro simulator and it was periodically analyzed their fate along the whole transit. Mucin agar carriers replaced the intestinal mucus layer and a basic feed medium represented the intestinal lumen contents. A three-day in vitro study was performed using microbiota from the pooled faeces of healthy individuals that were stabilized simulating colon conditions. The MRSA population survived the decreasing gastric pH levels unharmed, but it was affected by the organic acids produced by the enteric microbiota along the transit into the simulator. It was, in fact, no longer viable after 24 h of incubation with luminal colon microbiota, whereas counts of 4 log cfu/g were still obtained in the mucin agar carriers after 72 h of incubation. Despite the ability of MRSA to overcome human stomach acidic conditions, these results confirm the hypothesis that competitive microbiota may control MRSA intestinal colonization.

Orientador: Claudio Alexandre Gobatto
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Educação Física
Made available in DSpace on 2018-08-27T03:03:23Z (GMT). No. of bitstreams: 1 MassellidosReis_IvanGustavo_D.pdf: 3926036 bytes, checksum: 3eda6aa62a61d88c274a00563e5b2943 (MD5) Previous issue date: 2015
Resumo: Sabe-se que o estresse físico exerce uma função moduladora na expressão gênica dos MCTs 1 e 4 por meio de vias de sinalização moleculares aparentemente distintas envolvendo o co-ativador-1 'alfa' do receptor gama ativado por proliferador do peroxissomo (PGC-1?) e subunidade 1 'alfa' do fator induzível por hipóxia (HIF-1?) respectivamente. Apenas uma única sessão de exercício de resistência (endurance) está associada ao aumento na expressão do MCT1 e PGC-1?, mas não do MCT4, no músculo esquelético vasto lateral de humanos, enquanto o exercício intermitente de alta intensidade parece afetar ambos MCTs 1 e 4 além do PGC-1?. No entanto pouco se conhece sobre o efeito simultâneo do estresse físico sobre o HIF-1?, MCts 1 e 4 e PGC-1? em diferentes tecidos e tipos de fibra. É provável que tanto a expressão quanto a transcrição dos co-ativadores e fatores de transcrição envolvidos na modulação dos MCTs 1 e 4 frente ao estresse físico sejam afetadas pelas características da atividade e ainda variem de acordo com o tipo e especificidade do tecido analisado. Dessa forma, o objetivo desse estudo foi verificar o efeito agudo de uma única sessão de natação até exaustão ou de 30 minutos contínuos ou 25 minutos acumulados intermitentemente, em uma intensidade equivalente ou 20% superior a máxima fase estável de lactato, sobre a expressão gênica e conteúdo protéico dos HIF-1?, MCTs 1 e 4, PGC-1?, imediatamente, 2, 4 e 8 horas após a sessão de exercício, em tecidos chaves para metabolismo do lactato (fibras esqueléticas I e II, fígado, coração) de ratos. O exercício físico aumenta a expressão proteica e mRNA em relação ao grupo controle para maior parte dos genes que foram analisados, porém, não há diferenças entre os grupos exercitados independente do tecido e do protocolo utilizado. Com exceção do tecido hepático cuja apenas a expressão de PGC-1? mRNA é estimulada, uma única sessão de exercício induz diferentes respostas ao longo de 8 horas na expressão mRNA e conteúdo de HIF-1?, MCTs 1 e 4, PGC-1?. Uma sessão contínua de volume reduzido ou uma sessão intermitente em intensidade 20% superior a MFEL, resultam nas mesmas adaptações de uma sessão contínua de 30 minutos de duração em intensidade equivalente a MFEL
Abstract: It is known that physical stress plays a role on regulating the gene expression of MCTs 1 and 4 by distinct molecular signaling pathways involving the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1?) and hypoxia inducible factor 1 alpha subunit (HIF-1?) respectively. Only a single endurance session is associated with increased expression of both PGC-1? and MCT1, but not of the MCT4 in the muscle vastus lateralis of humans, while the intermittent exercise of high intensity seems to affect, besides the PGC-1?, both MCTs 1 and 4. However, the knowledge about the simultaneously effect of the exercise stress on the HIF-1?, MCTs 1 and 4 and PGC-1? in different types of tissues and skeletal muscles is unknow. Probably, the transcription factors and the coativators involved in the exercise induced modulation of MCTs 1 and 4 can being differently affected by the exercise intensity and may vary according to the type and metabolic specificity of the tissue. Thus, the aim of this study was to investigate the acute effect of a single swimming session of 30 continuous minutes or 25 minutes accumulated intermittently or until exhaustion in the intensity equivalent or 20% higher than the maximum lactate steady state (MLSS), on the gene expression and protein content of HIF-1?, MCTs 1 and 4, PGC-1?, immediately, 2, 4 and 8 hours after, in key tissues to the lactate metabolism (skeletal muscle of type I and II, liver, heart) of rats. Physical exercise increased protein content and mRNA expression for most of the analyzed genes, however, there are no differences between the exercised groups independently of the tissue or protocol used. With the exception to liver, where only PGC-1? mRNA was stimulated, a single exercise bout induced different responses throughout 8 hours on mRNA expression and content of HIF-1?, MCTs 1 and 4, PGC-1?. Both, continuous or intermittent exercise, of reduced volume and in higher intensity (20%) results in similar responses of a continuous session of 30 minutes duration in the MLSS intensity
Doutorado
Biodinamica do Movimento e Esporte
Doutor em Educação Física

The gastrointestinal tract (GIT) regulates nutrient uptake, secretes hormones and has a crucial gut flora and enteric nervous system. Of relevance for these functions are the G protein-coupled receptors (GPCRs) and the solute carriers (SLCs). The Adhesion GPCR subfamily is known to mediate neural development and immune system functioning, whereas SLCs transport e.g. amino acids, fatty acids (FAs) and drugs over membranes. We aimed to comprehensively characterize Adhesion GPCR and SLC gene expression along the rat GIT. Using qPCR we measured expression of 78 SLCs as well as all 30 Adhesion GPCRs in a twelve-segment GIT model. 21 of the Adhesion GPCRs had a widespread (≥5 segments) or ubiquitous (≥11 segments) expression. Restricted expression patterns were characteristic for most group VII members. Of the SLCs, we found the majority (56 %) of these transcripts to be expressed in all GIT segments. SLCs were predominantly found in the absorption-responsible gut regions. Both Adhesion GPCRs and SLCs were widely expressed in the rat GIT, suggesting important roles. The distribution of Adhesion GPCRs defines them as a potential pharmacological target. FAs constitute an important energy source and have been implicated in the worldwide obesity increase. FAs and their ratios – indices for activities of e.g. the desaturase enzymes SCD-1 (SCD-16, 16:1n-7/16:0), D6D (18:3n-6/18:2n-6) and D5D (20:4n-6/20:3n-6) – have been associated with e.g. overall mortality and BMI. We examined whether differences in FAs and their indices in five lipid fractions contributed to obesity susceptibility in rats fed a high fat diet (HFD), and the associations of desaturase indices between lipid fractions in animals on different diets. We found that on a HFD, obesity-prone (OP) rats had a higher SCD-16 index and a lower linoleic acid (LA) proportions in subcutaneous adipose tissue (SAT) than obesity-resistant rats. Desaturase indices were significantly correlated between many of the lipid fractions. The higher SCD-16 may indicate higher SCD-1 activity in SAT in OP rats, and combined with lower LA proportions may provide novel insights into HFD-induced obesity. The associations between desaturase indices show that plasma measurements can serve as proxies for some lipid fractions, but the correlations seem to be affected by diet and weight gain.

Nucleic acid-based vaccines such as viral vectors, plasmid DNA and mRNA have been developed as a means to address limitations of both live attenuated and subunit vaccines. Among them, Self Amplifying mRNA vaccine (SAM) has been widely evaluated in different animal models and has been confirmed to be well tolerated and able to drive in vitro antigen expression. However, the molecular mechanism of action of SAM approach has not been fully elucidated. To address this gap, we employed a quantitative mass spectrometry (MS) approach to investigate the molecular fate of vaccine antigens encoded by SAM, from RNA delivery, until MHC-peptide presentation. In this work, we investigated the quantitative correlation between the antigen expression and epitope presentation on MHC class I molecules in a dose-range and time-lapse assay using myoblast cell cultures. Two delivery systems were compared, viral replicon particles (VRPs) and lipid nanoparticle, both already successfully tested in vivo with many different vaccine candidates. The data obtained show that the rate of intracellular antigen expression driven by VRPs is faster compared to the expression driven by SAM encapsulated in LNPs. Moreover we observed a tight correlation between the onset of protein expression and MHC class I epitope presentation for both delivery systems, providing strong evidence that epitope presentation is temporally linked to antigen translation. Furthermore, after detection, no evident differences in the intracellular amount of protein antigen and in the level of epitope peptide were observed, assuming that the main difference between VRPs and LNPs is only related to the mechanism of cellular uptake. Then we applied this technology to quantify the SAM encoded antigen in the muscle and lymph nodes of vaccinated mice at the site of injection, where this new type of vaccine are able to generate amount of antigen lower compare to the standard dose given by classical vaccines. Moreover, we move to an in vitro model of co-culture dendritic cells through, we were able to definitively confirm that this cells are not directly transfected by SAM but are able to up take antigen encoded by transfected myoblast cells. In this study we demonstrated the powerful use of Mass Spectrometry to better understand the mechanism of action of new kind of vaccines during the immune response.

Cellular events that rely on translation regulation has been well established, however the molecular details of factors involved in bringing translation control remains inadequately explored. RNA binding proteins form an integral part of transcriptional and post-transcriptional gene regulation pathway. However, the principles that govern the activity of an RNA binding protein is poorly explored. In this thesis, a systematic investigation has been done to delineate the contribution of individual RRM domains and arginine methylation in the RGG motif of an RNA binding protein, Sbp1 towards its function. Chapter 3 demonstrated that role of arginine methylation of Sbp1 RGG motif towards its translation repression and decapping enhancing activity. Pull-down assays indicated that Sbp1 interaction with eIF4G1 decreases when the methylating enzyme, Hmt1 is absent or the Sbp1 RGG motif is deleted. We also learned that Sbp1 mono-methylation increases upon glucose starvation stress, which is known to cause global translation repression in yeast. Moreover, arginine methylation of Sbp1 was found to be crucial for driving decapping activators such as Dhh1 and Scd6 to RNA granules. Together, our results have established functional relevance of arginine methylation towards translation repression and decapping enhancing ability of RNA binding protein, Sbp1. Chapter 4 investigated the role of RRM domains of Sbp1 towards causing over-expression mediated growth defect and localizing to RNA granules. RRM domains are the most abundant RNA binding domain that harbor 6-8 amino acid consensus sequence involved in RNA binding. Our results have demonstrated that upon deleting both the RRM domains and not the RNP sequence, SBP1 over-expression mediated growth defect can be rescued. Moreover, ∆RRM 1+2 mutant of Sbp1 could not localize to RNA granules upon glucose starvation than wild-type. These observations suggest that Sbp1 RRM domains function via sequences outside the RNP motif, which is yet to be discovered. Chapter 5 describes novel genetic interaction of SBP1 with genes involved in Non-sense mediated mRNA decay (NMD) pathway. Over-expression mediated growth defect by SBP1 is augmented upon individual deletion of UPF1, UPF2 and UPF3. However, the augmented growth phenotype was not due to an increase in the protein level of Sbp1. Our study has established a link between translation repression and mRNA decay. To summarize, our study has identified: i) importance of arginine methylation of Sbp1 in regulating its function, ii) contribution of RRM domains of Sbp1 in causing over-expression mediated growth defect and localize to RNA granules upon stress and iii) genetic modulators of Sbp1 function. These studies have been done in budding yeast. Although, yeast does not display tissue level specificity observed in complex organisms, the molecular pathways are largely conserved. Principles that govern mRNA fate in yeast can form the basis for hypothesizing how certain factors might function in humans.

Indiana University-Purdue University Indianapolis (IUPUI)
In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.