Academic literature on the topic 'MRNA fate'

The poly(A) tail of mRNA has an important influence on the dynamics of gene expression. On one hand, it promotes enhanced mRNA stability to allow production of the protein, even after inactivation of transcription. On the other hand, shortening of the poly(A) tail (deadenylation) slows down translation of the mRNA, or prevents it entirely, by inducing mRNA decay. Thus deadenylation plays a crucial role in the post-transcriptional regulation of gene expression, deciding the fate of individual mRNAs. It acts both in basal mRNA turnover, as well as in temporally and spatially regulated translation and decay of specific mRNAs. In the present paper, we discuss mRNA deadenylation in eukaryotes, focusing on the main deadenylase, the Ccr4–Not complex, including its composition, regulation and functional roles.

Translational Control of Gene Expression edited by N. Sonenberg, J. W. B. Hershey and M. B. Matthews Cold Spring Harbor Laboratory Press (2000) 1020 pages. ISBN 0–87969-568-4 US$115 At the beginning of the 90s most molecular biologists were focusing on transcription and RNA splicing. mRNA translation and its temporal and spatial regulation seemed research topics for insiders at that time. However, all aspects of mRNA fate in the cytoplasm will certainly attract much more attention during the next decade. The field is now flourishing with connections to all disciplines of biology. This book will help you to realize the tremendous variation of translational regulatory mechanisms existing in nature. The evidence for their importance has become so overwhelming that nobody seriously interested in gene expression can ignore it any longer. It is the great merit of the editors of this book that they have brought together an impressive series of first-class reviews written by the most prominent scientists in the field. The new monograph takes a fresh look at the field and is greatly expanded compared with the earlier 1996 version. The book is judiciously divided into two parts. The first part comprises eight broad chapters, giving an overview of the main principles of protein synthesis and its regulation. They serve as a thorough basis for the second part, which comprises twenty-eight chapters, each about 20 pages in length, that present in depth additional exciting areas in which there is strong research activity. Your appetite for this book will be stimulated right at the beginning by the wonderful introductory chapter, which is written jointly by the editors and defines the field in its entire complexity. Given that translation is of course a unifying principle of all living organisms, why are there such a large number of different control mechanisms modulating the use of mRNA templates and making actual protein level not predictable from RNA quantity alone? Are these just remnants of an RNA world or, as the authors seem to believe, effective adaptations for fine-tuning gene expression that have been opportunistically added during evolution? Five broad chapters are devoted to our knowledge of initiation, elongation and termination of translation both in eukaryotes and in prokaryotes. It is amazing how much detail has been added, in just the past five years, to our picture of the biochemistry, structure and function of ribosomes, initiation sites, and translation factors. However, translational control of gene expression is not just a matter of the translation machinery alone. It seems rather that the tremendously versatile mRNA sequences and structures impose the way they are seen by the translation apparatus and its factors. Particularly in eukaryotes, the untranslated parts of mRNAs play a decisive role by providing additional interaction sites for cytoplasmic proteins that modulate mRNA stability, mRNA localization or accessibility of mRNAs to translation. In turn, many of the proteins interacting with mRNA are themselves regulated by metabolites or post-translational modifications. This is beautifully documented in an exciting chapter on the role of translational control in developmental decisions. For example, in Drosophila, a specific cascade of factors acting on RNA localization and translation controls the anterior-posterior body axis. In C. elegans, the fate of germ-line cells is determined by translational repression. And you will find many more such examples. Another important section of the book is devoted to changes in translation that occur during virus infection. Again one is amazed by the variety of ways by which viruses divert the host translation apparatus for their own sake. The shorter chapters give insight into additional exciting areas in the field. For example, research into how heat shock or signal transduction pathways feed into translation, what we know about mRNA degradation of normal and nonsense-containing transcripts, and the evidence that local synaptic protein synthesis represents a molecular hallmark of learning and memory. This book is the most complete and up-to-date review of translational control mechanisms. It is a must for students entering the field, and it will constitute for many years a major reference guide for any investigator who is seriously interested in the full picture of gene expression.

Microarray studies in Saccharomyces cerevisiae have set the benchmark for genome-wide analyses, available data-sets covering practically every stage of gene expression from DNA-binding by transcription factors to mRNA export, sub-cellular localisation, translation and decay. A theme to emerge from such data has been the prevalence of coordinate gene regulation. Thus, gene modules or ?regulons? are well recognised at the level of gene transcription and the activity of transcription factors provides an obvious molecular explanation for such coordination. More surprising was the organisation of mRNAs into co-regulated ?post-transcriptional operons?. RNA-binding proteins (RBPs), but also ribonucleoprotein (RNP) complexes involving noncoding RNA, have been proposed as the conceptual equivalent of transcription factors at this level.

An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein–mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.

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.

Cellulitis is an acute, spreading infection of the deep dermis and subcutaneous fat characterized by erythema, warmth, swelling, and pain. Erysipelas is an infection of the more superficial cutaneous lymphatics and skin, including the epidermis as well as the upper reticular and papillary dermal layers. It is commonly located on the lower extremities, face, and ears. Cutaneous abscesses are purulent collections that occupy the dermis and deeper soft tissues. Effective treatment of cellulitis and erysipelas includes antibiotics and elevation of the affected area to assist in lymphatic drainage. Methicillin-resistant Staphylococcus aureus (MRSA) infection should be considered in all cellulitis or erysipelas patients who have failed initial therapy, have a history of or risk factors for MRSA, or have severe infection or systemic toxicity. The first-line treatment for a cutaneous abscess is incision and drainage (I&D); no data demonstrates the utility of antibiotic therapy in conjunction with I&D.

64-year-old woman with a history of fevers and anemia Coronal SSFSE (Figure 16.18.1) and fat-suppressed SSFP (Figure 16.18.2) images demonstrate a heterogeneous mass expanding the intrahepatic IVC. Coronal source image from venous phase acquisition of a 3D CE MRA (...

Microinjection of genetic components and dye into organotypic slices provides excellent single cell resolution for unraveling biological complexities, but is extremely difficult and time consuming to perform manually resulting in low yield and low use in the developmental biology field. We developed a computer vision guided platform to inject specimen with mRNA, and/or dye and investigated the efficiency of the process using organotypic slices of the mouse developing neocortex. We demonstrate that the system significantly increases yield of injection relative to manual use by an order of magnitude, allows for cell tracking over 0, 24, and 48 hours post injection in culture, and enables mRNA translation of injected product. The autoinjector platform thus can open the door to new types of experiments including investigating effects of mRNA concentration, and composition on cell fate, and tracking these effects on cell reprogramming and lineage.

Introdução: Os microRNAs são uma importante classe de pequenos RNAs endógenos não codantes. Esses pequenos RNAs (~ 22 nucleotídeos) agem como guia para um complexo ribonucleoprotéico clivar ou inibir o RNA mensageiro, atuando, desta forma, no silenciamento gênico pós-transcricional. Objetivo: O presente trabalho visou realizar uma breve revisão acerca da importância dos miRNAs no desenvolvimento vegetal. Material e Métodos: Desta forma, foi realizado um levantamento bibliográfico nas plataformas Google Scholar, Periódico CAPES, PubMed, Scielo e em livros acadêmicos. As palavras-chave utilizadas foram: Plant microRNAs, Plant microRNAs biogenesis, microRNAs in Plant development. De acordo com o levantamento os genes de miRNAs em plantas estão localizados, predominantemente, nas regiões intergênicas e são transcritos em um microRNA primário de fita dupla (pri-miRNA). Resultados: O pri-miRNA é processado por um complexo enzimático contendo diversas enzimas, entre elas a Dicer-Like1 (DCL1), originando um microRNA precursor (pre-miRNA) que também é processado no núcleo pelo complexo DCL1. Nessa etapa, a proteína Hua-Ehancer1 promove a metilação na hidroxila do carbono 2’ no último nucleotídeo, sendo essa modificação uma proteção contra a degradação química do miRNA. Com o auxílio da proteína Hasty (HST) o miRNA é transportado para o citoplasma e incorporado à proteína Argonauta (AGO1) formando o complexo RISC o qual poderá atuar clivando ou inibindo o mRNA, impedindo sua tradução. Com o avanço do sequenciamento de próxima geração (NGS) os estudos de transcriptomas permitiram a elucidação dos mecanismos de ação dos microRNAs, sendo possível a compreensão desde sua biogênese, regulação da abundância e interação com o mRNA. Conclusão: Os microRNAs possuem papel fundamental na embriogênese, organogênese, nas rotas metabólicas dos vegetais; são de extrema importância no controle de estresses bióticos e abióticos e atuam na transição da fase vegetativa para a reprodutiva, no desenvolvimento radicular, foliar, de flores e frutos, entre outros. O entendimento do modo de ação desses pequenos RNAs nos tecidos vegetais é de grande relevância para os programas de melhoramento vegetal que tem como princípio a biotecnologia e a biologia molecular.

Introdução: Com a pandemia do novo coronavírus, SARS-CoV2, a vacina tornou-se importante ferramenta no combate aos casos mais graves da doença. No entanto, essa foi alvo de diversas “fake news” divulgadas a seu respeito. Nesse contexto, uma das notícias falsas mais compartilhadas diz respeito à vacina Pfizer/BioNTech contra a COVID-19 que, por ser feita a partir de RNA, seria capaz de modificar o DNA de quem a recebe. Objetivos: Refutar sobre as inverdades compartilhadas sobre a vacina Pfizer/BioNTech ser capaz de mudar o DNA dos indivíduos que são vacinados. Metodologia: Foi realizada uma revisão bibliográfica a partir da avaliação de 9 periódicos publicados entre 2011 e 2021. Destes, foram selecionados seis (6), na base de dados PubMed e Scielo, utilizando como termos de pesquisa: “COVID-19 Vaccines ” e “Pandemic”, terminologias de acordo com o sistema de Descritores em Ciências da Saúde (DeCs). Resultados: A Pfizer, junto à BioNTech, foi a indústria farmacêutica pioneira no contexto de desenvolvimento de uma vacina que usa a plataforma de RNA mensageiro. Tal inovação científica gerou desconfiança e receio ao redor do globo, especialmente devido ao compartilhamento de informações falsas que afirmavam que o RNA seria capaz de modificar o DNA do vacinado. No entanto, vale ressaltar que o RNA usado na vacina é encapsulado em uma microesfera de lipídio e injetado no músculo. Ao entrar na célula, ele é encaminhado para o citoplasma, mas não chega a atingir a membrana do núcleo, local onde está o DNA. Dessa forma, ainda no citoplasma, a célula reconhece por meio se seus ribossomos o RNA mensageiro oriundo da vacina e, rapidamente, uma proteína é sintetizada. Em seguida, o RNA é degenerado, logo deixa de ser funcional, o que refuta a ideia de que ele é capaz de promover mutações no DNA. Conclusão: Portanto, as vacinas que se apoiam no mRNA não promovem alterações do núcleo celular. Assim, é necessário que os profissionais de saúde e a mídia tomem medidas para auxiliar o público a identificar o discurso por trás das “fake news”, além de evidenciar a importância de averiguar a informação recebida antes de compartilhá-la.

Bile acids are significant physiological factors for digestion, solubilization, absorption, toxic metabolites and xenobiotics. In addition, bile acids are responsible of signal transduction as well as metabolic regulation that activate several receptors such as farnesoid X receptor (FXR) and the membrane G-protein receptor 5 (TGR5). Activation of TGR5 by bile acids is associated with prevention of obesity as well as ameliorating the resistance to insulin via increasing energy expenditure. The objective of this research is to investigate TGR5 gene expression level in different fat depots including visceral or epididymal adipose tissue (eWAT), brown adipose tissue and inguinal adipose tissue (iWAT) and to study the response of TGR5 gene expression to the antiobesity treatment (SFN). Three groups of male CD1 mice were used in this study; lean group fed with SCD, DIO mice on HFD and DIO obese mice treated with anti-obesity treatment. Body weight (BW) and phenotype data were evaluated by weekly including blood samples for analysis of glucose, insulin, leptin, triglycerides (TG). Total RNA was extracted from different fat depots and RT-PCR profiler array technology was used to in order to assess the mRNA expression of TGR5 and leptin. There was significant downregulation of TGR5 gene expression level in obese (DIO) mice and remarkable upregulation of TGR5 gene expression after successful weight loss in DIO mice treated with SFN in time dependent manner at 1 weeks and 4 weeks of ip applications. In conclusion, obesity is associated with decrease in expression of TGR5 in different fat depots and treatment with anti-obesity drug (Sulforaphane) causes stepwise upregulation of TGR5 gene expression in epididymal white adipose tissue parallel stepwise decrease in body weight. Increase of expression of TGR5 in DIO mice in eWAT is accompanied by improvement in glucose homeostasis and insulin action.

Obesity is a growing problem worldwide, and recent data indicated that 20% of the populations would be obese. Obesity arises as a multifactorial disease caused by inherited traits that interact with lifestyle factors such as diet and physical activity. The liver plays an essential role in the gluco-regulation via regulating glucose, lipid and protein metabolism. The process of glucose metabolism is controlled by a range of molecular mechanisms and genes which affect the metabolism of the liver during intake of high fat diet (HFD). The objective of this research is to investigate the profile of hepatic gene expression of glucose metabolism in mice on HFD treated with leptin (5 mg/kg BW Ip injection). Ten wild type CD1 mice fed on HFD is used for this study, where groups are control (vehicle - leptin) and test group (vehicle + leptin). Body weight (BW) was measured, and blood chemistry, insulin and leptin were measured at the end of the experiments. Total RNA was isolated from the liver tissue, and RTPCR profiler array technology was used to evaluate the mRNA expression of 84 essential genes of hepatic glucose metabolism. The data of the BW and blood chemistry are not significantly different between the two groups. Leptin treatment enhanced the metabolic pathways and the candidate genes of the different metabolic pathway; glycogen metabolism such as Gys1, Gys2 and Pygm, pentose phosphate shunt such as Rpia and suppressed the glycolysis such as Aldob, and TCA cycle such as Mdh1b. In conclusion, this study has shown that leptin could affect the profile of the hepatic mouse genes of glucose metabolism in the early stages of HFD to induce obesity

The shoot apical meristem establishes plant architecture by continuously producing new lateral organs such as leaves, axillary meristems and flowers throughout the plant life cycle. This unique capacity is achieved by a group of self-renewing pluripotent stem cells that give rise to founder cells, which can differentiate into multiple cell and tissue types in response to environmental and developmental cues. Cell fate specification at the shoot apical meristem is programmed primarily by transcription factors acting in a complex gene regulatory network. In this project we proposed to provide significant understanding of meristem maintenance and cell fate specification by studying four transcription factors acting at the meristem. Our original aim was to identify the direct target genes of WUS, STM, KNAT6 and CNA transcription factor in a genome wide scale and the manner by which they regulate their targets. Our goal was to integrate this data into a regulatory model of cell fate specification in the SAM and to identify key genes within the model for further study. We have generated transgenic plants carrying the four TF with two different tags and preformed chromatin Immunoprecipitation (ChIP) assay to identify the TF direct target genes. Due to unforeseen obstacles we have been delayed in achieving this aim but hope to accomplish it soon. Using the GR inducible system, genetic approach and transcriptome analysis [mRNA-seq] we provided a new look at meristem activity and its regulation of morphogenesis and phyllotaxy and propose a coherent framework for the role of many factors acting in meristem development and maintenance. We provided evidence for 3 different mechanisms for the regulation of WUS expression, DNA methylation, a second receptor pathway - the ERECTA receptor and the CNA TF that negatively regulates WUS expression in its own domain, the Organizing Center. We found that once the WUS expression level surpasses a certain threshold it alters cell identity at the periphery of the inflorescence meristem from floral meristem to carpel fate [FM]. When WUS expression highly elevated in the FM, the meristem turn into indeterminate. We showed that WUS activate cytokinine, inhibit auxin response and represses the genes required for root identity fate and that gradual increase in WUCHEL activity leads to gradual meristem enlargement that affect phyllotaxis. We also propose a model in which the direction of WUS domain expansion laterally or upward affects meristem structure differently. We preformed mRNA-seq on meristems with different size and structure followed by k-means clustering and identified groups of genes that are expressed in specific domains at the meristem. We will integrate this data with the ChIP-seq of the 4 TF to add another layer to the genetic network regulating meristem activity.

Both the genes and cDNA sequences encoding the b-subunits of black carp LH and FSH were isolated, cloned and sequenced. Sequence analysis of the bcFSHb and LHb5'flanking regions revealed that the promoter region of both genes contains canonical TATA sequences, 30 bp and 17 bp upstream of the transcription start site of FSHb and LHb genes, respectively. In addition, they include several sequences of cis-acting motifs, required for inducible and tissue-specific transcriptional regulation: the gonadotropin-specific element (GSE), GnRH responsive element (GRE), half sites of estrogen and androgen response elements, cAMP response element, and AP1. Several methods have been employed by the Israeli team to purify the recombinant b subunits (EtOH precipitation, gel filtration and lentil lectin). While the final objective to produce pure recombinantGtH subunits has not yet been achieved, we have covered much ground towards this goal. The black carp ovary showed a gradual increase in both mass and oocyte diameter. First postvitellogenic oocytes were found in 5 yr old fish. At this age, the testes already contained spermatozoa. The circulating LH levels increased from 0.5 ng/ml in 4 yr old fish to >5ng/ml in 5 yr old fish. In vivo challenge experiments in black carp showed the initial LH response of the pituitary to GnRH in 4 yr old fish. The response was further augmented in 5 yr old fish. The increase in estradiol level in response to gonadotropic stimulation was first noted in 4 yr old fish but this response was much stronger in the following year. In vivo experiments on the FSHb and LHb mRNA levels in response to GnRH were carried out on common carp as a model for synchronom spawning cyprinids. These experiments showed the prevalence of FSHP in maturing fish while LHP mRNA was prevalent in mature fish, especially in females. The gonadal fat-pad was found to originate from the retroperitoneal mesoderm and not from the genital ridge, thus differing from that reported in certain amphibians This tissue possibly serves as the major source of sex steroids in the immature black carp. However, such a function is taken over by the developing gonads in 4 yr old fish. In the striped bass, we described the ontogeny of the neuro-endocrine parameters along the brain-pituitary-gonadal axis during the first four years of life, throughout gonadal development and the onset of puberty. We also described the responsiveness of the reproductive axis to long-term hormonal manipulations at various stages of gonadal development. Most males reached complete sexual maturity during the first year of life. Puberty was initiated during the third year of life in most females, but this first reproductive cycle did not lead to the acquisition of full sexual maturity. This finding indicates that more than one reproductive cycle may be required before adulthood is reached. Out of the three native GnRHs present in striped bass, only sbGnRH and cGnRH II increased concomitantly with the progress of gonadal development and the onset of puberty. This finding, together with data on GtH synthesis and release, suggests that while sbGnRH and cGnRH II may be involved in the regulation of puberty in striped bass, these neuropeptides are not limiting factors to the onset of puberty. Plasma LH levels remained low in all fish, suggesting that LH plays only a minor role in early gonadal development. This hypothesis was further supported by the finding that experimentally elevated plasma LH levels did not result in the induction of complete ovarian and testicular development. The acquisition of complete puberty in 4 yr old females was associated with a rise in the mRNA levels of all GtH subunit genes, including a 218-fold increase in the mRNA levels of bFSH. mRNA levels of the a and PLH subunits increased only 11- and 8-fold, respectively. Although data on plasma FSH levels are unavailable, the dramatic increase in bFSH mRNA suggests a pivotal role for this hormone in regulating the onset and completion of puberty in striped bass. The hormonal regulation of the onset of puberty and of GtH synthesis and release was studied by chronic administration of testosterone (T) and/or an analog of gonadotropin-releasing hormone (G). Sustained administration of T+G increased the mRNA levels of the PLH subunit to the values characteristic of sexually mature fish, and also increased the plasma levels of LH. However, these changes did not result in the acceleration of sexual maturation. The mRNA levels of the bFSH subunit were slightly stimulated, but remained about 1/10 of the values characteristic of sexually mature fish. It is concluded that the stimulation of FSH gene expression and release does not lead to the acceleration of sexual maturity, and that the failure to sufficiently stimulate the bFSH subunit gene expression may underlie the inability of the treatments to advance sexual maturity. Consequently, FSH is suggested to be the key hormone to the initiation and completion of puberty in striped bass. Future efforts to induce precocious puberty in striped bass should focus on understanding the regulation of FSH synthesis and release and on developing technologies to induce these processes. Definite formulation of hormonal manipulation to advance puberty in the striped bass and the black carp seems to be premature at this stage. However, the project has already yielded a great number of experimental tools of DNA technology, slow-release systems and endocrine information on the process of puberty. These systems and certain protocols have been already utilized successfully to advance maturation in other fish (e.g. grey mullet) and will form a base for further study on fish puberty.