Thèses sur le sujet « Exogenous and endogenous attention »

The studies presented in this thesis investigated the neural correlates of attention in touch. In particular, the electrophysiology of exogenous tactile processing and inhibition of return (IOR) - an area previously unexplored. In all studies a variation of the Posner cue-target paradigm was used. Typically, a cue was presented to the left or right hand. Following a stimulus onset asynchrony of 800 ms, a target would appear at the same or opposite hand. Behavioural results consistently demonstrated IOR when employing a simple target detection task, showing that IOR is a reliable phenomenon in touch. The concurrently recorded event related potentials (ERPs) demonstrated an early attention modulation of the N80 in all studies presented in this thesis, regardless of the presence or absence of IOR. This early component likely reflects processing of the exogenous lateralized cues. Following the N80, the attention modulations varied across studies. The conclusion to be drawn from this thesis is that not one particular ERP component is directly associated with IOR. Analysis of endogenous tactile attention (Chapter V) demonstrated modulations at the N140 and Nd components. Moreover, correlation analysis showed that larger ERP attention modulation was associated with a larger behavioural effect, demonstrating a novel relationship between ERP modulations and response time effects. Analysis of the cue-target interval has previously only been investigated during endogenous orienting. Here, and for the first time, an anterior directing attention negativity (ADAN) was demonstrated during exogenous orienting. This ADAN was unaffected by varying posture suggesting exogenous tactile attention and IOR are somatotopically coded. Indications of an external frame of reference were only demonstrated during shifts of endogenous attention, as indicated by the presence of a late directing attention positivity (LDAP) (endogenous counter-predictive task presented in Chapter V). The final study of this thesis (Chapter VI) demonstrated that varying visual perceptual load influenced tactile processing. Specifically, high perceptual load led to elimination of IOR. Moreover, the P100 for irrelevant tactile stimuli was significantly reduced in high versus low load condition. This suggests perceptual load may suppress irrelevant tactile stimuli relatively early (around 100 ms post stimuli onset) during tactile processing. Taken together, this thesis presents a series of experiments which map out effects of endogenous and exogenous attention and how these mechanisms interact, both through behaviour and underlying neural correlates.

The experiments in this thesis were designed to examine the consequences of endogenous and exogenous spatial cueing in a dual-task set-up. The first experiments, presented in Chapters 2 and 3, explored whether spatial attention generalises across dimensions in the same location. Chapters 4, 5, and 6 contain a second series of experiments using exogenous cues, in which cue properties were manipulated. A dual-task set-up was used in all studies in this thesis, with a display of four random dot kinematograms containing motion and colour features. In order to examine whether endogenous attention may be spatially oriented to only one feature dimension, a central cue was presented that was 70% valid for the location of only one task. Both tasks showed validity effects, but the task for which the cue was informative showed larger attentional modulation. This suggests that spatial attention is not a single 'spotlight' but can be biased in favour of expected features. There was also asymmetry in the tasks, whereby the validity effect was modulated for motion, but comparable for colour regardless of the task for which the cue was informative. This asymmetry was also evident when using uninformative exogenous cues preceding the same tasks. Peripheral luminance and colour cues affected the validity effects for the motion and colour tasks differently, suggesting that the relationship between cue properties and proceeding stimuli modulates attentional effects. The size of a frame cue leads to different attentional effects on tasks of different sizes. These experiments make a considerable contribution to the spatial attention literature, by showing that spatial attention may be biased either by cue properties or cue information, suggesting that spatial attention and feature-based attention may interact. They also provide further evidence that motion is better represented than colour in visual attention.

Converging evidences from neurophysiological, neuropsychological and functional neuroimaging literature suggested the involvement of a large-scale fronto-parietal network in the volitional and reflexive attentional control, which enables the biological systems to select relevant portions of the input stream for facilitating processing, in order to flexibly guide behavior on the basis of internal or external goals. This system acts in tight interplay with lateralized structures deputed to the achievement and maintenance of an adequate level of arousal, and with medial frontal structures responsible for the on-line modification of action and thought in response to an ever-changing external world. This project aims at clarifying the spatio-temporal dynamics of the recruitment of these three tightly related systems (alertness, orienting and executive control) and at a better explanation of integrative effects previously reported, by means of a series of high-density electrophysiological recording experiments combined with source localization methods. Experiment 1 combined a spatial cueing paradigm with a flanker task, showing early occipital and parietal activations in response to valid cues, and a lateralized pattern of sources for the responses of the phasic alertness system to both spatially valid and spatially neutral cues; additionally, we reported behavioral, electrophysiological and functional indexes of a modulation exerted by the orienting system upon the executive control one. Experiment 2 compared lateralized shifts of attention elicited by centrally and peripherally presented spatially valid and neutral cues, in a combined cueing and go/no-go task. The results again suggested an early involvement of the selective attention system in response to the task-relevant cues, as expressed in occipito-temporal enhanced responses to the valid cues as compared to the neutral ones; additionally, lateralized superior parietal and frontal activations were recorded, concurrently with attentional shifts. Again, a modulation of the conflict monitoring/response inhibition system was observed in case of validly cued stimuli, as indexed by anticipations of the fronto-central NoGoN2 in response to non-targets preceded by valid warning signals. Experiment 3 tested the hypothesis of bidirectional modulations between the attention and control systems, by means of an integrated stop-signal/flanker task. The results showed that the selective response inhibition, required in case of incompatible flankers in the primary stimuli, interfered with the attentional switch towards the relevant stop-signals, as reflected in dimmed auditory responses to the stop-tones. All in all, the present results point toward a complex interplay among the three systems, and suggest that any interpretation of the scientific results obtained in paradigms addressing the attentional networks should account also for alertness and executive control effects, which are indirectly manipulated when capitalizing on cueing/switching paradigms.

1. Introduction The multidisciplinary approach of using brain signals for directly controlling external devices, like computers or prosthesis, is named brain-computer interface (BCI). Farwell and Donchin (1988) showed that it is possible for humans to communicate using a BCI, by means of their event-related potentials (ERPs; e.g., P300), without the involvement of their voluntary muscle activity. The use of brain-wave-guided BCIs offered new perspectives regarding communication and control of devices for patients suffering from severe motor impairment or for patients who completely paralyzed, such as the patients affected by amyotrophic lateral sclerosis (ALS), in the latest stages of the illness. In the last two decades an important scientific and clinical challenge has been the development of efficient BCIs for ALS patients. Most of the progress in the field has been mainly concerned with algorithm improvement for better signal classification. In contrast, only few studies have addressed, to date, the role of cognitive mechanisms underlying the elicitation of brain-signals in BCIs. In the present study we investigated the possibility to modulate the brain signal and, by doing so, the performance of an ERP-guided BCI system, by designing and implementing three new interfaces in which participants were required to perform covert visuospatial attention orienting (Posner, 1980), in order to control the movement of a cursor on a monitor. 2. Experiment 1 The effects of covert visuospatial attention orienting within an ERP-guided BCI were tested on healthy participants. We compared the effectiveness of three visual interfaces, each of whom elicited different modalities of covert visuospatial attention orienting (exogenous vs. endogenous). Twelve adult participants performed 20 sessions, using the abovementioned ERP-guided BCI interfaces to control the movement of a cursor. Brain waves were recorded on each trial and were subsequently classified online, by means of an ad-hoc algorithm. Each time the target ERPs were correctly classified, the cursor moved towards the target position. The “endogenous” interface was associated with significantly higher performance than the other two interfaces during the testing sessions, but not in the follow-up sessions. Endogenous visuospatial attention orienting can be effectively implemented to increase the performance of ERP-guided BCIs. 3. Experiment 2 To investigate whether the findings reported in Experiment 1 depended on the used classification system, we performed an offline reclassification of the data of Experiment 1. The online analysis of the epochs was made via Independent Component Analysis (ICA), which, in turn, was followed by fixed features extraction and support vector machine (SVM) classification. The offline epochs analysis was performed by means of a genetic algorithm (GA), which permitted us to retrieve the relevant features of the signal to be classified, and then to categorise them with a logistic classifier. The offline analysis confirmed the advantages derived from the use of the “endogenous” interface. The performance-related findings were in line with those obtained in the neurophysiological data analysis. Nonetheless, epoch categorization was performed better with the GA algorithm than with the ICA: the higher mean and the smaller standard deviation of the classification performed with the GA seem to promise a possible improvement of the ERP-guided BCI also on online tests. 4. Experiment 3 On the basis of the results of Experiments 1 and 2, we tested the efficacy of two visual interfaces, each of whom elicited different modalities of covert visuospatial attention orienting (exogenous vs. endogenous), in ALS patients. Ten ALS patients performed 16 online sessions with each interface. Although the ALS patients had a performance of about 70% with both the interfaces, the endogenous interface elicited a larger difference on ERP potentials between target and non-target trials. These results supported the hypothesis that the use of the endogenous interface may offer a more efficient channel of communication for ALS patients with respect to the use of the exogenous interface. 5. Conclusions Neurological diseases that affect the motor system may impair communication abilities of patients, as in the case of amyotrophic lateral sclerosis. This pathology might evolve in the locked-in syndrome (LIS), a condition in which patients remain conscious but cannot move any of their muscles. For instance, they may become unable to express their opinions and decisions on important questions regarding their clinical treatment or their living and biological wills. The BCIs represent a potential solution to the communication problems of ALS-LIS patients. Despite the fact that more than 20 years have passed since the first published article on a P300-guided BCI, the effects of cognitive mechanisms (i.e., executive functions, attention, memory, etc.) involved in brain signal elicitation have not been investigated extensively. In the abovementioned experiments, we tested the effect of covert visuospatial attention orienting on an ERP-guided BCI, by comparing a number of visual interfaces, each of whom elicited a different modality of covert visuospatial attention orienting. Taken together, the results supported our main hypothesis: It is possible to modulate the performance of an ERP-guided BCI, by using endogenous or exogenous visuospatial attention orienting. Of particular relevance is the fact that our ALS patients were able to use endogenous visuospatial attention orienting and, by doing so, they could increase their performance in an ERP-guided BCI. We suggest that the study of covert visuospatial attention orienting is essential for developing efficient visual BCIs for patients who cannot control their eye movements. Implementing principles taken from cognitive psychology, may improve BCIs efficiency. This, in turn, can increase the benefits for patients with severe motor and communication disabilities. Finally, an efficient cognitive-based BCI may have the considerable ethical implication of “giving a voice” to CLIS-ALS patients
1. Introduzione Farwell e Donchin (1988) per primi hanno dimostrato la possibilità che l’uomo ha di comunicare usando i potenziali evento correlati (ERP; e.g., P300), senza bisogno di usare alcun muscolo per tale fine. Questa scoperta ha offerto nuove prospettive per la comunicazione ed il controllo di periferiche in pazienti affetti da gravi disabilità motorie o completamente paralizzati, come nel caso dei pazienti affetti da sclerosi laterale amiotrofica (SLA), negli stadi più avanzati di malattia. L’approccio multidisciplinare che consente di tradurre segnali cerebrali direttamente in comandi per controllare computer o protesi meccaniche è chiamato brain-computer interface (BCI). Negli ultimi vent’anni un’importante sfida scientifica è stata quella di sviluppare una BCI efficace, affinché potesse essere usata nella pratica clinica con i pazienti. I progressi più rilevanti fatti finora riguardano principalmente la registrazione e l’elaborazione dei segnali cerebrali, grazie ad algoritmi sempre più potenti ed efficaci nella categorizzazione dei biosegnali. Minore attenzione è stata posta, invece, nell’investigare il ruolo dei meccanismi cognitivi che sottendono l’uso di una BCI. Nel presente studio è stata indagata la potenzialità dei partecipanti di modulare specifiche onde cerebrali e, di conseguenza, l’efficacia di un sistema BCI guidato dagli ERP, attraverso l’uso di diversi processi di orientamento implicito dell’attenzione visuospaziale (Posner, 1980). A tale scopo sono state progettate e testate tre nuove interfacce visive per controllare il movimento di un cursore su un monitor. 2. Esperimento 1 Nel primo esperimento è stato testato l’effetto dell’orientamento implicito dell’attenzione visuospaziale in partecipanti sani, il cui scopo era di controllare il movimento di un cursore con una BCI guidata da ERP, per raggiungere specifici bersagli. È stato confrontato l’uso di tre interfacce, ciascuna delle quali prevedeva l’utilizzo di una specifica modalità dell’orientamento implicito dell’attenzione visuospaziale (esogeno vs. endogeno). Dodici partecipanti adulti hanno eseguito 20 sessioni, con ciascuna delle tre interfacce. Simultaneamente, gli ERP associati a ciascun trial di ogni interfaccia erano registrati e classificati da un algoritmo ad hoc. Ogni volta che gli ERP associati alla direzione della posizione bersaglio erano correttamente classificati, il cursore era mosso di un passo verso la posizione bersaglio. I partecipanti hanno ottenuto un’accuratezza migliore nel controllo del cursore con l’interfaccia che prevedeva l’orientamento endogeno dell’attenzione visuospaziale rispetto alle due interfacce che prevedevano l’orientamento esogeno. 3. Esperimento 2 Nel secondo studio è stata eseguita una classificazione offline degli ERP registrati nell’Esperimento 1, con lo scopo di verificare se gli effetti ottenuti nell’Esperimento 1 fossero indipendenti dal tipo di algoritmo di classificazione utilizzato. La classificazione online dei segnali cerebrali avveniva attraverso l’analisi delle componenti indipendenti (ICA), un’estrazione di 78 caratteristiche stabilite a priori del segnale, e la loro categorizzazione attraverso un algoritmo matematico di tipo lineare (support vector macchine: SVM). La riclassificazione offline è stata eseguita per mezzo di un algoritmo genetico (genetic algorithm: GA), che rilevava ad personam le caratteristiche significative del segnale, le quali, infine, venivano categorizzate attraverso un classificatore logistico. Il metodo di classificazione offline nell’Esperimento 2 ha confermato l’effetto ottenuto nell’Esperimento 1. Questi risultati sono stati confermati anche dalle analisi statistiche eseguite sui dati neurofisiologici. Inoltre, le medie di accuratezza più alte e la minore variabilità associate al sistema di classificazione offline sembrano offrire potenziali miglioramenti dell’efficacia dell’uso in tempo reale della nostra BCI. 4. Esperimento 3 Alla luce dei risultati riportati negli Esperimenti 1 e 2, è stata testata l’efficacia di un’interfaccia che prevedeva l’uso dell’orientamento esogeno dell’attenzione visuospaziale e di un’altra che prevedeva l’uso dell’orientamento endogeno, con pazienti affetti da SLA. Dieci pazienti con SLA hanno eseguito 16 sessioni con ciascuna delle due interfacce. Anche se i pazienti hanno ottenuto un’accuratezza di circa 70% con entrambe le interfacce, è stata registrata una maggior differenza tra gli ERP target e quelli non-target con l’uso dell’interfaccia “endogena”. Questi risultati supportano l’ipotesi che l’interfaccia che usa l’orientamento endogeno dell’attenzione visuospaziale consenta un miglior controllo del sistema BCI, con conseguenti vantaggi comunicativi per i pazienti affetti da SLA. 5. Conclusioni Le patologie neurologiche che colpiscono il sistema motorio possono intaccare i normali canali di comunicazione, come nel caso di pazienti affetti dal SLA. Questa malattia può sfociare nello stato denominato sindrome locked-in (LIS), una condizione clinica in cui i pazienti sono completamente paralizzati ma mantengono intatta la loro consapevolezza. Nella condizione di LIS, un paziente non può comunicare, non potendo così esprimere la propria opinione riguardo alle scelte etico-giuridiche legate alla sua condizione clinica. Le BCI rappresentano una potenziale soluzione ai problemi comunicativi dei pazienti nella LIS. Negli ultimi vent’anni di ricerca scientifica sulle BCI è stata rivolta grande attenzione alle componenti tecnologiche implicate nella registrazione del segnale cerebrale e nella sua classificazione in comandi per controllare specifiche periferiche. Viceversa, minor attenzione è stata posta alle caratteristiche dell’utente nell’utilizzo delle BCI, in particolar modo riguardo alle componenti cognitive coinvolte. Negli esperimenti riportati nella presente tesi, abbiamo testato l’efficacia di diverse interfacce, ciascuna delle quali utilizzava una specifica modalità dell’orientamento implicito dell’attenzione visuospaziale (endogena o esogena). I risultati di questi esperimenti supportano l’ipotesi che è possibile modulare l’efficacia di una BCI guidata da ERP attraverso l’implementazione di interfacce visive che utilizzano diversi principi dell’orientamento implicito dell’attenzione visuospaziale. Tale risultato è di particolare rilevanza dal punto di vista clinico per i pazienti affetti da SLA, negli stadi terminali di malattia, cioè quando entrano nella condizione clinica di LIS. In particolare nell’Esperimento 3 è riportato come l’ampiezza degli ERP sia diversamente modulata nelle due interfacce testate e questo fatto può giocare un ruolo rilevante nello sviluppo di un efficace sistema BCI che permetta la comunicazione a pazienti affetti da SLA nella condizione di completa LIS. I nostri risultati portano evidenze di come l’implementazione dei principi della psicologia cognitiva nello sviluppo di una BCI ne possano modulare l’efficacia, e questo a vantaggio dei pazienti affetti da gravi disabilità motorie. In conclusione, un’efficace applicazione dei principi cognitivi nello sviluppo delle BCI può avere l’effetto rilevante di “dare una voce” a pazienti in stato di completa LIS

Cette thèse portait sur l'attention visuelle sélective, dans le domaine non-spatial, c'est-à-dire la capacité à favoriser le traitement perceptif de certains objets de la scène visuelle au détriment des autres. En particulier, l'objectif était d'étudier, à l'aide de tâches de recherche visuelle, les différentes interactions possibles entre les processus endogènes (liés aux intentions et connaissances du sujet) et les processus exogènes (liés aux caractéristiques perceptives des objets). Une première série d'expériences démontrait que la saillance pouvait présenter des effets relativement durables. Cependant, ces effets de saillance pouvaient être favorisés ou contrecarrés par des processus endogènes (induits par indiçage portant sur la taille), et modulés par l'amorçage perceptif. Une seconde série d'expériences démontrait que la résistance à l'interférence induite par un distracteur saillant dépendait de ressources attentionnelles centrales, et était modulée par la charge perceptive. Enfin, la troisième série d'expériences validait l'hypothèse d'une intégration entre des signaux endogènes et exogènes compatibles mais strictement distincts. La falsification de l'inégalité de Miller (1982) précisait que cette intégration reposait sur une coactivation effective, non sur une simple facilitation statistique entre signaux strictement indépendants. Le locus cognitif de cette intégration semble être le système perceptif. L'ensemble des données présentées souligne la flexibilité du contrôle attentionnel, et invite à développer un modèle général de l'attention visuelle sélective basé sur l'hypothèse de compétition biaisée (Desimone & Duncan, 1995)
This thesis was about visual selective attention, toward non-spatial features, that is, the ability to favour the perceptual processing of some objects in the visual scene, at the expense of others. In particular, the aim was to study, through visual search experiments, the various possible interactions between endogenous (linked with incentives and knowledge of subjects) and exogenous (linked with perceptual properties of the objects) processes. The first series of experiments showed long-lived salience effects. These salience effects could, hewever, be favoured or overriden by endogenous processes (induced by cueing the target size), and modulated by perceptual priming. The second series evidenced that resisting the interference induced by a salient distractor depended on central attentional resources, and was modulatted by perceptual load. Finally, the third series demonstrated that separate endogenous and exogenous signals could genuinely integrate. This integration could not be accounted for by a race between strictly independant signals, as evidenced by the falsification of the Miller's (1982) inequality. This integration might occur in the perceptual system. Theglobal set of data highlighted the flexibility of endogenous attentional control, and invited developping a general model of visual selective attention, on the basis of the biased competition hypothesis

Individuals with high levels of plasma triglycerides are at high risk to develop cardiovascular disease (CVD), currently one of the major causes of death worldwide. Recent epidemiological studies show that loss-of-function mutations in the APOC3 gene lower plasma triglyceride levels and reduce the incidence of coronary artery disease. The APOC3 gene encodes for apolipoprotein (APO) C3, known as an inhibitor of lipoprotein lipase (LPL) activity. Similarly, a common gain-of-function mutation in the LPL gene is associated with reduced risk for CVD. LPL is central for the metabolism of lipids in blood. The enzyme acts at the endothelial surface of the capillary bed where it hydrolyzes triglycerides in circulating triglyceride-rich lipoproteins (TRLs) and thereby allows uptake of fatty acids in adjacent tissues. LPL activity has to be rapidly modulated to adapt to the metabolic demands of different tissues. The current view is that LPL is constitutively expressed and that the rapid modulation of the enzymatic activity occurs by some different controller proteins. Angiopoietin-like protein 4 (ANGPTL4) is one of the main candidates for control of LPL activity. ANGPTL4 causes irreversible inactivation through dissociation of the active LPL dimer to inactive monomers. Other proteins that have effects on LPL activity are the APOCs which are surface components of the substrate TRLs. APOC2 is a well-known LPL co-factor, whereas APOC1 and APOC3 independently inhibit LPL activity. Given the important role of LPL for triglyceride homeostasis in blood, the aim of this thesis was to find small molecules that could increase LPL activity and serve as lead compounds in future drug discovery efforts. Another aim was to investigate the molecular mechanisms for how APOC1 and APOC3 inhibit LPL activity. Using a small molecule screening library we have identified small molecules that can protect LPL from inactivation by ANGPTL4 during incubations in vitro. Following a structure-activity relationship study we have synthesized lead compounds that more efficiently protect LPL from inactivation by ANGPTL4 in vitro and also have dramatic triglyceride-lowering properties in vivo. In a separate study we show that low concentrations of fatty acids possess the ability to prevent inactivation of LPL by ANGPTL4 under in vitro conditions. With regard to APOC1 and APOC3 we demonstrate that when bound to TRLs, these apolipoproteins prevent binding of LPL to the lipid/water interface. This results in decreased lipolysis and in an increased susceptibility of LPL to inactivation by ANGPTL4. We demonstrate that hydrophobic amino acid residues that are centrally located in the APOC3 molecule are critical for attachment of this protein to lipid emulsion particles and consequently for inhibition of LPL activity. In summary, this work has identified a lead compound that protects LPL from inactivation by ANGPTL4 in vitro and lowers triglycerides in vivo. In addition, we propose a molecular mechanism for inhibition of LPL activity by APOC1 and APOC3.

Despite the vast research on the social bias in decision-making, relatively little is known about biases in voting behaviour. The main aim of this research was to explore alternative indirect methods to observe biases in decision-making and voting behaviour. A proximity bias was first observed in the rather unusual setting of the Weakest Link TV game show, when contestants avoided casting negative votes against their closest neighbours. This proximity bias was most profound for the contestant closest to the voter. Two field experiments were designed to test whether this Neighbour Effect occurred in different social contexts, among the first-year undergraduate students. The first study asked first-year undergraduate students in a lecture (n=449) to vote for another person seated in the same row. The same Neighbour Effect occurred when the vote carried a nasty (negative) outcome for the recipient however, when the vote valence changed to a nice (positive) outcome the Neighbour Effect disappeared. In negative voting, the result of the field experiment confirmed the original observation in the Weakest Link. However, a reverse polarity voting pattern was also found in the positive voting. This suggests participants significantly favoured their closest neighbour(s). The second field experiment used Prisoner’s Dilemma with undergraduates in a lecture theatre (n= 229) to test the Neighbour Effect. The undergraduates played the game with another player seated in the same row and in the same block in a lecture theatre. The results showed a neighbour effect because the players were significantly more likely to cooperate with a neighbour that a non-neighbour. To conclude the findings from this study suggested that the Neighbour Effect is a robust bias in strategic decision-making and voting.

Retroviruses must integrate their genome into the host DNA as a necessary step of their replication cycle. Normally, retroviruses integrate into somatic cells and are transmitted, from infected to uninfected hosts, as “exogenous” retroviruses. On rare occasions, they can infect germ line cells and become part of the host genome as “endogenous” retroviruses (ERVs), which are transmitted vertically to the offspring and inherited as Mendelian genes. During evolution, most ERVs have accumulated mutations that rendered them defective and unable to produce infectious viral particles. Some ERVs, however, have maintained intact open reading frames for some of their genes, and have been co-opted by the host as they fulfil important biological functions. Sheep betaretroviruses represent a unique model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) co-exists with the highly related endogenous JSRVs (enJSRVs). The sheep genome harbours at least twenty-seven enJSRV loci and, most likely, the process of endogenization is still occurring. During evolution, one of these enJSRV loci, enJS56A1, has acquired a defective and transdominant Gag polyprotein that blocks the late replication steps of related retroviruses, by a mechanism known as JSRV late restriction (JLR). Interestingly, enJSRV-26, a provirus that integrated in the sheep germ line less than two hundred years ago, possesses the unique ability to escape JLR. In this thesis, the molecular basis of JLR escape was investigated. The main determinant of JLR escape was identified in the signal peptide of enJSRV-26 envelope protein (SP26). A single amino acid substitution in SP26 was found to be responsible for altering its intracellular localization as well as its function as a post-transcriptional regulator of viral gene expression. Interestingly, interference assays demonstrated that enJSRV-26 relies on the presence of the functional signal peptide of enJS56A1 envelope protein (SP56) in order to escape JLR. In addition, the ratio between enJSRV-26 and enJS56A1 Gag polyproteins was found to be critical to elude JLR. Finally, sequence analyses revealed that the domestic sheep has acquired, by genome amplification, several copies of the enJS56A1 provirus, reinforcing the hypothesis that this locus has provided an evolutionary advantage to the host. This study unveils critical aspects of JLR that were previously unknown, and provides new insights on the molecular mechanisms governing the interplay between endogenous and exogenous sheep betaretroviruses.

Retroelements are some of the most successful parasites studied because of their ability to reverse transcribe and permanently integrate into the host genome. Host cells have, therefore, evolved multiple control mechanisms, such as cellular restriction factors, to protect their genomes from the pathogenic and mutagenic effects of retroelements. Identification of the full complement of these proteins is vital to comprehend the capacity of the host to regulate these genetic parasites. Human MOV10 is a putative RNA helicase with inhibitory or stimulatory roles in the replication of several RNA viruses, and the homologs of which play vital roles in the restriction of viruses and endogenous retroelements. Furthermore, MOV10 interacts with antiviral APOBEC3 proteins and core post-transcriptional RNA silencing machinery, all of which colocalise in cytoplasmic mRNA processing bodies and stress granules. Considering MOV10 cellular associations and homolog functions, the capacity of MOV10 to regulate the replication of a diverse panel of genetically distinct retroelements was investigated here. Ectopically overexpressed MOV10 potently restricts the replication of retroviruses as well as the propagation of LTR and non-LTR endogenous retroelements. Significantly, RNAi-mediated silencing of endogenous MOV10 enhances the replication of endogenous retroelements, but not exogenous retroviruses demonstrating that natural levels of MOV10 suppress retrotransposition. MOV10 overexpression decreases the level of HIV-1 genomic RNA packaged into nascent virions and also impacts the accumulation of reverse transcription products in target cells. The molecular mechanism/s by which MOV10 inhibits retroelements remains unclear, however, the anti-retroelement activities of MOV10 and APOBEC3 proteins are independent. Moreover, MOV10 is not essential for miRNA-mediated translation repression or slicer activity in cultured cells. In sum, ectopically overexpressed human MOV10 inhibits divergent exogenous and endogenous retroelements and, more significantly, the capacity of endogenous MOV10 to specifically suppress retrotransposition highlights it as a potential restriction factor of human retrotransposons in somatic cells.

Atherosclerotic and restenotic lesions develop as a result of an excess inflammatory response to vascular injury. Glucocorticoid hormones have widely-recognised anti-inflammatory and anti-proliferative properties which appear to make them ideal candidates for inhibition of vascular lesion development. Indeed, administration of glucocorticoids to experimental animals does inhibit the growth of vascular lesions in some models. In addition, glucocorticoids are currently being trialled clinically as anti-restenotic agents. However, glucocorticoid excess in patients, either as a result of Cushing’s syndrome or chronic steroid therapy, is associated with enhanced CVD risk. Therefore, the effects of glucocorticoids on vascular lesion development remain imperfectly understood. The overall objective of these studies was to explore the influence of endogenous and exogenous glucocorticoids on vascular lesion development using murine models of atherosclerosis (ApoE-/- mice fed a “western” diet) and neointimal hyperplasia (wireinduced femoral artery injury). The work described in this thesis addresses the hypothesis that glucocorticoids are pro-atherogenic, yet anti-restenotic. Mice were bilaterally adrenalectomised to investigate the role of endogenous glucocorticoids on vascular lesion development. Removal of the adrenal glands had no influence on atherogenesis or neointima development. The influence of exogenous glucocorticoids on lesion development was assessed by orally administering dexamethasone (0.1 or 0.8mg/kg/day). Atherosclerotic lesion burden was augmented by dexamethasone administration. Conversely, fibro-proliferative neointimal proliferation was inhibited by dexamethasone. However, this effect was obscured by thrombotic lesion development. It was proposed that this thrombotic effect is attributable to increased plasminogen activator inhibitor-1 (PAI-1), which was tested using PAI-1 deficient mice. Although PAI-1 was found to mediate the systemic pro-thrombotic effect of dexamethasone, it is not required for the enhanced development of thrombotic lesions at the site of intra-luminal injury. These results suggest that physiological levels of endogenous glucocorticoids play a limited role in vascular lesion development. Conversely, although exogenous glucocorticoids inhibit fibro-proliferative intimal hyperplasia, they appear to have significant detrimental influences on lesion development, augmenting atherosclerosis and inducing thrombotic neointimal lesion formation following vascular injury. Further research is therefore required to improve the cardiovascular outcome of patients requiring glucocorticoid therapy and for the use of glucocorticoids as antirestenotic agents.

Microbiology and Immunology
Ph.D.
Systemic lupus erythematosus (SLE) is complex autoimmune disease in which autoantibodies form against double stranded DNA (dsDNA) and nuclear antigens. Autoantigen immune complexes form, deposit in the vasculature, and cause multisystem organ damage. Both genetic and environmental factors contribute to the development of SLE. This thesis will explore three major themes found in the study of SLE: 1) Bacterial infection as an environmental trigger, 2) cytokine dysregulation in immune cells, and 3) the treatment of end organ damage in the form of lupus nephritis. Viral infections have long been associated with the development of systemic autoimmune disease, but the mechanisms by which chronic bacterial infections may promote autoimmunity remain unclear. In chapter three we show that a component of bacterial biofilms, the amyloid-like protein “curli”, irreversibly forms fibers with bacterial or eukaryotic DNA during biofilm formation. This interaction accelerates amyloid polymerization and creates potent immunogenic complexes that activate immune cells, including dendritic cells, to produce cytokines such as type I interferons, which are pathogenic in SLE. When given systemically, curli/DNA composites trigger immune activation and production of autoantibodies in lupus-prone and wild type mice. We also found that infection with curli-producing bacteria triggered higher autoantibody titers in lupus-prone mice compared to curli-deficient bacteria. These data provide a mechanism by which the microbiome and biofilm-producing enteric infections may contribute to the progression of SLE and point to a potential molecular target for treatment of autoimmunity. Cytokine dysregulation is also common in SLE patients. Serum cytokines are often elevated during active disease, including type I IFNs and IL-10. In chapter four we demonstrate that Il10 is a type I IFN response gene and has increased basal expression in dendritic cells (DCs) derived from pre-disease lupus-prone Sle1,2,3 mice. We show that Sle1,2,3-derived DCs overproduce IL-10 in response to TLR ligands and that this is the result of autocrine signaling though the type I IFN receptor (IFNAR). These results suggest that dysregulation of cytokine signaling in the myeloid compartment may contribute to IL-10 dysregulation in SLE. Renal disease remains a major cause of morbidity and mortality in SLE. A number of mouse models of chronic kidney disease have implicated the EGFR-family receptors in the progression of renal fibrosis and dysfunction. In chapter five we show that renal expression of ErbB2 is increased in murine lupus. We therefore asked if EGFR-family inhibition could prevent murine lupus nephritis. To test this possibility we used lapatinib, an EGFR-ErbB2 dual kinase inhibitor, in an IFN-accelerated model of murine lupus. We found that lapatinib administration lowered autoantibody levels but worsened renal disease. Lapatinib failure to treat murine lupus nephritis despite lowered autoantibody levels suggests EGFR-family signaling is required for tissue repair in the acute phase of kidney injury. Together this thesis clearly demonstrates the complexity of systemic autoimmune disease – bringing us to the crossroads of immunity and tolerance. The combination of both environmental triggers (e.g. bacterial infection) and genetic susceptibility (e.g. intrinsic cytokine dysregulation) leads to end organ damage (e.g. lupus nephritis). Here we sought to explore each aspect of disease progression in the hopes to develop better interventions for systemic autoimmune disease.
Temple University--Theses

In this thesis, two-echelon systems with exogenous and endogenous lead times are studied for the orders placed by the retailer(s) from the supplier. The retailer(s) employ periodic-review base-stock policy, namely (R,S) policy. For the case the demand during review period is i.i.d. and the probability distribution is Normal for each review period, a new method is proposed for exogenous lead time case under stationary policy. The results of the proposed method is then compared with the results of the existing methods in the literature and it is concluded that the proposed method provides service levels sufficiently close to target levels whereas the existing methods do not necessarily provide target levels. We use the simulation to study the endogenous replenishment lead time case. The proposed method is modified when the retailer employs stationary policy and it is seen that the proposed method gives no-stockout probabilities close to target levels. Moreover, the impacts of using adaptive policy on the performance of the retailer are studied for endogenous replenishment lead time case. It is concluded that updating of the order-up-to-level deteriorates the performance of the retailer. Finally, it is questioned whether it is beneficial for a retailer to use adaptive policy in a supply chain with two retailers. Simulation results show that the deterioration in the performance of the retailer handling stationary policy is larger compared to the other retailer handling adaptive policy and the deteriorations get larger in the case of an increase in update frequency or in utilization of the supplier.

Abstract Melatonin is an important modulator of several physiological and behavioural processes, and it influences the function of many different tissues. Melatonin has effective antioxidative properties, but some of its actions in mammals are also mediated through the MT1 and MT2 melatonin receptors. Antioxidative properties are seen especially when the melatonin concentration is high (≥ nM), and melatonin's affinity for its receptors appears at lower concentrations (pM). Recently, the involvement of melatonin in protecting the heart against cardiac diseases, including myocardial infarction (MI), has been brought out. MI alters the structure and function of myocardium, attenuating for example cardiac contractility by affecting the amount and function of the essential Ca2+ handling proteins, dihydropyridine receptor (DHPR), ryanodine receptor (RyR2) and sarco-endoplasmic reticulum (SR) Ca2+-ATPase2 (SERCA2). MI also evokes many adaptive responses in organisms, such as elevated production of atrial and brain natriuretic peptides (ANP and BNP). In this thesis, the expression of MT1 and MT2 receptor mRNAs was investigated in several rat tissues. Furthermore, the effect of MI and exogenous melatonin on the rat endogenous melatonin and on the expression of cardiac MT1, MT2, DHPR, RyR2 and SERCA2 proteins was evaluated. The concentrations of ANP and BNP were also measured after post-MI melatonin administration. The results show the expression of MT1 and/or MT2 receptor mRNAs in the hypothalamus, retina, small intestine, liver and heart, which indicates that at least some effects of melatonin could be mediated through the receptors in these tissues. Melatonin synthesis in the pineal gland increased rapidly in response to MI, supporting an important role of endogenous melatonin in protecting the heart after MI. Furthermore, exogenous melatonin altered the mRNA expression of DHPR, RyR2 and SERCA2 after MI, suggesting that melatonin might contribute to the post-infarction cardiac contractile function. The results also revealed a novel, positive relationship between melatonin and ANP, and thereby bring out one more possible way of melatonin to protect the heart against MI-induced injuries. Taken together, the present thesis (i) supports the notion that melatonin is an important endogenous protective agent of the organism, and (ii) extends our knowledge of melatonin's post-infarction cardioprotective actions
Tiivistelmä Melatoniini osallistuu monien fysiologisten toimintojen ja käyttäytymisen säätelyyn sekä vaikuttaa useiden eri kudosten toimintaan. Melatoniini on tehokas antioksidantti, mutta jotkut sen vaikutuksista välittyvät myös MT1 ja MT2 melatoniini reseptorien kautta. Antioksidatiiviset vaikutukset tulevat esiin erityisesti silloin, kun melatoniinin pitoisuus on korkea (≥ nM). Alhaisemmilla pitoisuuksilla (pM) on puolestaan havaittavissa melatoniinin sitoutuminen reseptoreihinsa. Viime aikoina on tullut esille melatoniinin mahdollinen suojavaikutus sydänsairauksia, kuten sydäninfarkteja, vastaan. Sydäninfarkti muuttaa sydänlihaksen rakennetta ja toimintaa esimerkiksi vaikuttamalla supistuksen kannalta välttämättömien proteiinien, dihydropyridiini reseptorin (DHPR), ryanodiini reseptorin (RyR2) ja sarko-endoplasmakalvoston Ca2+-ATPaasi2:n (SERCA2) lukumääriin ja toimintaan, ja sitä kautta muun muassa heikentää sydämen supistuvuutta. Sydäninfarkti laukaisee elimistössä myös monia adaptiivisia vasteita, kuten eteispeptidin (ANP) ja aivojen natriureettisen peptidin (BNP) lisääntyneen erityksen. Tässä väitöstyössä tutkittiin MT1 ja MT2 reseptorien mRNAn ilmentymistä useissa rotan eri kudoksissa. Lisäksi tutkittiin sydäninfarktin ja eksogeenisen melatoniinin vaikutuksia rotan endogeeniseen melatoniiniin sekä sydämen MT1, MT2, DHPR, RyR2 ja SERCA2 proteiinien ekspressioon. Myös ANP ja BNP pitoisuudet mitattiin. Tulokset osoittivat MT1 ja/tai MT2 reseptori mRNAn ilmentymisen hypotalamuksessa, silmän verkkokalvolla, ohutsuolessa, maksassa ja sydämessä, minkä perusteella ainakin osa melatoniinin vaikutuksista saattaisi olla reseptorivälitteisiä näissä kudoksissa. Tulosten mukaan käpyrauhasen melatoniinisynteesi lisääntyi nopeasti sydäninfarktin jälkeen, mikä tukee käsitystä endogeenisen melatoniinin tärkeästä roolista infarktin jälkeisessä sydämen suojauksessa. Lisäksi eksogeeninen melatoniini muutti DHPR:n, RyR2:n ja SERCA2:n mRNA ekspressiota infarktin jälkeen, mikä voisi merkitä, että melatoniini saattaa vaikuttaa infarktin jälkeiseen sydämen supistuvuuteen. Tulosten osoittama positiivinen riippuvuus melatoniinin ja ANP:n välillä tuo puolestaan esille yhden uuden mahdollisen keinon, jonka kautta melatoniini voisi suojata sydäntä infarktin aiheuttamia vaurioita vastaan. Yhteenvetona voidaan todeta, että tämä väitöstyö (i) tukee käsitystä, että endogeenisella melatoniinilla on tärkeä merkitys elimistön suojaamisessa, ja (ii) laajentaa tietämystämme infarktin jälkeisistä melatoniinin sydäntä suojaavista vaikutuksista

The discovery of stem and progenitor cells capable of ongoing neurogenesis in the adult mammalian brain has raised hope that we will one day be able to harness their intrinsic regenerative capacity following injury. Development of such therapeutic strategies relies on a comprehensive understanding of the underlying regulation of the neurogenic process. To this end, I show, in this thesis, that cultured post-natal hippocampal neural progenitor cells (NPCs) express a specific repertoire of connexins (Cx), a family of channel forming proteins critical for communication prior to the development of functional chemical synapses. I show that this pattern of Cx expression, specifically Cx43 and Cx45, is modulated by interaction with the extracellular matrix component laminin providing evidence of extracellular matrix-cell interaction in the regulation of intrinsic Cx expression and function in postnatal NPCs. In adult brain, I show, for the first time, that Cx45 localizes to all cell types of the neuronal lineage with the exception of the type 3 doublecortin (DCX)-positive NPCs. Using a loss of function approach, I show that this expression is required for the normal proliferation of type 1 nestin and glial fibrillary acidic protein-positive stem like NPCs but not for the differentiation or survival of their progeny in the adult hippocampus. With respect to exogenous pharmacological cues that influence hippocampal neurogenesis, this thesis also demonstrates that chronic treatment with a sub-set of selective serotonin reuptake inhibitor antidepressants, fluoxetine and escitalopram, increases the proliferation but not the survival of adult NPCs in healthy, non-depressed mice. Further, standard post-operative analgesia with the opiate buprenorphine inhibits the proliferation of DCX-positive adult NPCs and increases the survival of their progeny. Finally, over the course of the research for this thesis, it became clear that exposing research animals to even very subtle environmental changes can influence the basal neurogenic process. Ultimately this work further highlights the exquisite sensitivity of the regulation of what is already recognized to be a highly dynamic process and provides important insight into the neurogenic process that can be used to inform future therapeutic development and application.

The focus here is on the log-normal version of the spatial interaction model. In this context, we consider spatial econometric specifications that can be used to accommodate two types of dependence scenarios, one involving endogenous interaction and the other exogenous interaction. These model specifications replace the conventional assumption of independence between origin-destination-flows with formal approaches that allow for two different types of spatial dependence in flow magnitudes. Endogenous interaction reflects situations where there is reaction to feedback regarding flow magnitudes from regions neighboring origin and destination regions. This type of interaction can be modeled using specifications proposed by LeSage and Pace (2008) who use spatial lags of the dependent variable to quantify the magnitude and extent of feedback effects, hence the term endogenous interaction. Exogenous interaction represents a situation where spillover arise from nearby (or perhaps even distant) regions, and these need to be taken into account when modeling observed variation in flows across the network of regions. In contrast to endogenous interaction, these contextual effects do not generate reaction to the spillovers, leading to a model specification that can be interpreted without considering changes in the long-run equilibrium state of the system of flows. We discuss issues pertaining to interpretation of estimates from these two types of model specification, and provide an empirical illustration. (authors' abstract)

Molecular magnetic resonance imaging (MRI) methods have the potential to provide detailed information regarding cellular and molecular processes at small scales within the human body. Nuclear signals from chemical samples can be probed using specialised MRI techniques, to highlight molecular contrast from particular enzymes or metabolites. The aim of the work described in this thesis is to investigate both exogenous and endogenous contrast mechanisms using fluorine MRI and chemical exchange saturation transfer (CEST) respectively, in order to detect molecular changes in vitro. Initial theoretical work investigates the factors which affect fluorine MRI signals and provides a theoretical framework to determine the sensitivity of such experiments. A novel paramagnetic fluorine sensor to detect enzyme activity is then characterised using high field nuclear magnetic resonance (NMR), showing 60 to 70–fold increases in T1 relaxation values upon enzyme interaction. The effects on the fluorine lineshape from varying sample temperature and solvent were investigated. The possibility of imaging is demonstrated, but further investigations using the theoretical framework found pre–clinical implementation of the sensor is limited by the achievable experimental sensitivity. Efforts then focussed on CEST molecular methods, which are not limited by sensitivity. A protocol is developed to target amide protons in an in vitro cancer cell model, with parameters optimised following simulation of the expected contrast. Analysis of CEST results were aided through use of a support vector machine (SVM) to distinguish group differences between cancer cells and control samples. A linear classifier was found to be suitable to discriminate between samples.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
This year it is estimated that over 34,000 American women will be diagnosed with triple-negative breast cancer (TNBC), an aggressive disease resistant to current targeted therapies. Consequently, development of new therapeutics that can be used to combat TNBC is an area of intense medical research. The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates many biological processes. Dysregulation of normal AhR expression and function is observed in breast cancers and promotes tumor growth. It is likely that AhR activation is involved in many steps of mammary tumor progression; however, the endogenous AhR ligand(s) driving these functions in mammary tissue remains a mystery. Here, we surveyed tryptophan metabolites to identify endogenous AhR agonists in mammary epithelial cells. Several metabolites, including 6-formylindolo[3,2-b]carbazole (FICZ), xanthurenic acid, indoxyl sulfate and kynurenic acid were found to activate the AhR in a mammary epithelial cell line and to bind directly to human AhR. Untargeted metabolomic studies identified indoxyl sulfate in cell extracts and strongly suggested that serum added to cultures is its source. The common presence of an efficacious AhR agonist in human sera and in serum used in our studies suggests the intriguing possibility that AhR activity in vivo is controlled, at least in part, by production of this bacteria-derived metabolite, potentially linking the microbiome to AhR-mediated mammalian cell function. Given the potential role of the AhR in mammary tumorigenesis, we sought to develop a new method for high throughput identification of novel AhR modulators for therapeutic applications. By screening of structure-guided chemical libraries, we identified CB7993113 as a pure, competitive AhR antagonist. In vivo, CB7993113 inhibited the acute toxicity associated with exposure to 7,12-dimethylbenz[α]anthracene, a prototypic AhR agonist. Additionally, CB7950998 was discovered to be a non-toxic AhR agonist that is able to decrease cell proliferation in human ER- breast cancer cells. These results are the first steps in the preclinical investigation for these AhR modulators. These studies advance the understanding of the endogenous agonists that drive endogenous AhR activation in mammary cells, and present two novel exogenous AhR ligands to be investigated for their therapeutic potential through modulation of AhR activity.

Regulators of G-protein signaling (RGS) proteins are a family of proteins that act as GTPase accelerating proteins (GAPs) through their interaction with GΑ subunits, including GΑo, GΑi, and GΑq but not GΑs. This increased rate of hydrolysis of GTP to GDP temporally regulates G-protein coupled receptor (GPCR) signaling. A member of this family, RGS4, has been implicated in several neurological disorders including Parkinson's Disease (PD). A hallmark of PD is the induction of oxidative stress within dopaminergic neurons. In this thesis, we evaluate the role of oxidative stress, including lipid peroxidation products with 4-hydroxy-2-nonenal (4HNE) as a model, in regulating RGS4 activity within neurons. Utilizing transfected RGS4, we evaluated whether RGS4 is readily modified by physiologically relevant concentrations of 4HNE by immuonoprecipitation of RGS4 from 4HNE treated cells. Further examination of recombinant RGS4 by mass spectrometry, revealed that RGS4 is readily modified at several cysteine residues by 4HNE, including C148. Modification at this residue has been shown to be a critical site for allosteric regulation of RGS4. This is confirmed through a malachite green based phosphate generation assay we developed to observe the GAP activity of RGS4 on its native binding partner GΑi. This malachite green based assay was then adapted for high throughput screening. The assay was successfully miniaturized to a 1536-well format. In a screen of 2300 compounds, 4 were identified as hits. The development of this simple and cheap assay can be adapted for usage with a variety of RGS proteins with little work to interrogate other pathways and identify novel RGS modulators. Finally, expansive study of PD has linked oxidative stress to the pathology of both diseases. What has not been discerned is the potential relationship between oxidative stress and the induction of RGS4. In support of the hypothesis, we evaluated the potential relationship between oxidative stress and RGS4 expression. This was accomplished by evaluating two striatal neuron like cell lines, SH-SY5Y and HCN-1A. After treatment with hydrogen peroxide, both cell lines showed increased RGS4 in response to oxidative stress. This response is not however related to mRNA expression, indicating this change is most likely an adjustment of proteasomal regulation of RGS4. This phenomenon may explain the rapid onset of Parkinsonian motor symptoms in reserpine treated animal models of PD, as excess dopamine in the cytoplasm may be rapidly metabolized in reactive products.

La gestione razionale di impianti moderni ed intensivi richiede un utilizzo delle risorse improntata alla massimizzazione degli assorbimenti ed al mantenimento degli equilibri morfologici e funzionali. Molte delle pratiche agronomiche vedono il loro effetto mediato dall’interazione con l’apparato radicale. Una conoscenza delle conseguenze morfologiche, architetturali, anatomiche e metaboliche che i fattori ambientali hanno sullo sviluppo radicale è pertanto imprescindibile ad un raggiungimento sostenibile degli obiettivi produttivi. L’azione di fattori esogeni si combina inevitabilmente con aspetti che riguardano la fenologia e gli equilibri interni alla singola pianta. La sfida, per l’acquisizione di un background efficace nella gestione della pianta, consiste nell’integrazione di influenze singole e nella definizione di modelli capaci di chiarire i rapporti di stimolo e competizione che si instaurano a livello radicale e determinano precise strategie di allocazione e utilizzo delle risorse. L’apparato radicale è preposto allo svolgimento di una vasta serie di funzioni e ciascuna di queste è prevalentemente attribuita a specifiche strutture. Alle radici di supporto si affianca la frazione di radici così dette “fini” a loro volta distinguibili in radici preposte al trasporto, all’esplorazione e allo stoccaggio di riserve (pioniere), e radici con funzione di assorbimento (fibrose). Diverso è il costo metabolico di ciascuna di queste strutture e diverso il ruolo giocato nel mantenimento degli equilibri della pianta. Uno degli obiettivi del lavoro è stato quello di valutare secondo quali principi la costituzione delle strutture radicali venisse variamente promossa a seconda delle condizioni endogene ed ambientali e quali fossero le conseguenze di queste scelte di allocazione di risorse sul metabolismo e sull’efficienza del sistema generato in risposta a stress. Integrando la frazione ipogea con quella epigea, il lavoro ha altresì esplorato le dinamiche di sinergia e competizione tra organi, alla base della periodicità e stagionalità dei comportamenti della radice. Identificati i principi alla base degli equilibri nella singola pianta, uno sforzo ulteriore è stato rappresentato dall’inserimento del sistema pianta all’interno di una fitocenosi e dalla valutazione di comportamenti radicali in relazione alle dinamiche di competizione/sinergia instaurate con altre specie vegetali. Il lavoro approccia in maniera iniziale lo studio delle dinamiche di relazione a livello ipogeo e si propone di analizzare le conseguenze morfologiche e metaboliche di una convivenza di specie senza porsi l’obiettivo di definire la natura e le cause di tali rapporti. Lo studio ha potuto dimostrare che per l’olivo: 1. Le basse temperature atmosferiche, fino ad un livello soglia di 7°C (temperatura minima), hanno indotto una riduzione lo sviluppo radicale. La relazione con le temperature aeree e quindi con l’attività della parte epigea è risultata più influente della temperatura del suolo stesso. Elevate temperature e scarsa disponibilità idrica sono state alla base di un ridotto tempo di emivita delle radici sottili, più longeve se prodotte in primavera che in estate ed a profondità maggiori piuttosto che superficialmente. 2. La ridotta disponibilità idrica ha influenzato lo sviluppo morfologico e la funzionalità della radice. In particolare, in presenza di condizioni non omogenee, è stata ridotta al minimo l’allocazione delle risorse in strutture pioniere posizionate in nicchie asciutte. La scarsa disponibilità idrica ha portato ad una riduzione dell’attività metabolica delle strutture radicali siano esse pioniere o assorbenti, ad un aumento del livello di danneggiamento delle pareti cellulari in strutture assorbenti e ad un accumulo di suberina e lignina nei tessuti soprattutto in strutture pioniere. 3. In condizioni non limitanti, lo sviluppo dei germogli non ha comportato una riduzione degli accrescimenti radicali ed il picco di maggior accrescimento dei due organi è stato, al contrario, ampiamente simultaneo. Lo sviluppo vegetativo è inoltre risultato essere positivamente correlato con l’aumento di produzione di ramificazioni laterali negli organi ipogei. 4. La condivisione dello spazio da parte di apparati di specie diversa ha ridotto lo sviluppo radicale sia in termini di accrescimenti medi per singola radice sia in termini di numero di elementi in attiva crescita. La competizione tra strutture erbacee ed arboree è risultata particolarmente forte nei primi 0.2 m di suolo mentre è andata attenuandosi più in profondità.
Modern and intensive orchards management requires a rational use of resources, aiming to maximize resources uptake and to maintain morphologic and functional balances. Many agronomic practices play a role which is mediated by the root system. A deep knowledge of environmental effects on root morphology, architecture, anatomy and metabolism is therefore necessary, in order to sustainably achieve the production target. Environmental factors effect is combined, on natural conditions, with plant phenology and internal physiological balances. Our goal, in order to achieve a background useful on plant management, was to integrate single factor influences and describe stimulus and competition relationships acting on root. We aimed to decipher dynamics determining resource use and allocation pattern. Root system cooperate to a wide set of functions, each of them is specifically carried out by precise root structures. Beside the main roots, the whole system is composed by roots so called “fine”. This last group may be profitably further divided into two main class: pioneer roots which are charged to soil exploration, resources transport, and reserves storage, and fibrous roots dedicated to nutrients and water absorption. Each of those two classes has a different metabolic cost and plays a different role in maintaining plant balances. One of the goals of the present work was to identify principles driving root building and consequences of resources allocation strategies on the whole system metabolism and efficiency in reaction to stress The current work also tried to clarify synergy and competition dynamics within organs as a fundament for root growth pattern seasonality and periodicity. Once clarified relationship for single plant, a further step was to insert the plant system in a phytocoenosis, in order to evaluate root behavior as influenced by interspecific relationships. The current work just basically approached the topic of ipogenous correlation and aimed to analyze morphological consequences of species coexisting without looking specifically for reasons inducing changes in growth pattern. The study showed that in olive trees: 1. Low air temperature, up to a threshold level of 7°C (minimum temperature), reduced root development during the winter. Relationship with air temperature, and therefore with canopy activity, resulted more influent than soil temperature in Mediterranean climate. High temperatures and low soil moisture reduced fine root half-life in the summer. Roots grew during spring lasted longer than those grew during summer, as well as roots that were formed deeper into the soil survived longer than the shallower ones. 2. Non-homogeneous reduced soil moisture in split pot experiments influenced root morphology and metabolism by drastically reducing resources allocation to pioneer structures. Low soil moisture also induced a reduction on root metabolism, either on pioneer or fibrous, it caused an increase of cells damage on fibrous roots and an increase on suberine and lignine concentration especially on pioneer root tissues. 3. In not-limiting conditions shoot growth did not inhibit root development and growth peak for the two organs was instead synchronous. Vegetative growth was furthermore positively correlated with increased lateral branching rate. 4. Sharing space with other species roots reduced the number of growing tips as well as the total root system growth. Competition was found to be more dramatic on the top soil, while grass effects where gradually overcome at a depth over 0.2 m.

The BBB is a biological firewall that carefully regulates the cerebral microenvironment by acting as a physical, metabolic and transport barrier to molecules bound for the brain. This selectively permeable interface was modelled in this thesis using the recently established immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate interactions with endogenously and exogenously derived molecules of clinical significance. The endogenous molecules in question are the cationic amino acids (CAA) L-arginine, the precursor for nitric oxide (NO), and asymmetric dimethylarginine (ADMA), an endogenously derived analogue of L-arginine that acts as a potent inhibitor of NO production. As well as being an important vasodilator, NO has regulatory roles in the brain and on the BBB itself. Transport mechanisms utilised by L-arginine are known, but are not fully understood for ADMA – particularly so at the BBB. This is of clinical significance giving the emerging role of ADMA in many brain and cerebrovascular diseases. Understanding these transport mechanisms and other interactions of ADMA with the BBB is therefore very important for the study of disease emergence, detection and progression. We discovered in the hCMEC/D3s that high concentrations of ADMA could induce endothelial dysfunction in a BBB permeability model, leading to an increase in paracellular permeability to the paracellular marker FITC-dextran (40kDa). We also illustrated interactions of ADMA with a variety transport proteins, basing the study design on our observed L-arginine interactions. The CAA transport system y+ was heavily implicated for both molecules through the use of established inhibitors. Furthermore, the expression of CAT-1, the best known protein from this group was confirmed in the hCMEC/D3s. We also found evidence of an efflux transport system for ADMA (but not Larginine), implicating the neutral and CAA transporter ATB0,+. The protein expression of ATB0,+ was also confirmed in the cells. Intracellular ADMA was even shown to induce transstimulation of extracellular L-arginine, providing evidence for a role of ADMA in the 'L-arginine paradox', a phenomenon observed in vivo that administered L-arginine can alleviate the effects of NO reduction (such as vasoconstriction), despite there being 20-30 times the amount of L-arginine present to saturate the NO producing enzyme. In summary, our endogenous molecule findings from this thesis identify the likely transport mechanisms used by ADMA and implicate ADMA in endothelial dysfunction as well as the ‘Larginine paradox’. These data are not only important with regards to the brain, but apply to other microvascular endothelia such as those found in peripheral cardiovascular system, where ADMA remains a major area of investigation. The exogenous molecules studied during this PhD are drugs currently used to treat the second-stage of human African Trypanosomiasis (HAT). HAT is a neglected parasitic disease that continues to persist in sub-Saharan Africa. It is fatal if untreated. Recently, it has been described that eflornithine and nifurtimox combination therapy (NECT), improves the efficacy of both drugs compared to their monotherapy, although why this happens remains unclear. We hypothesised that it may be due to improved CNS delivery, although we failed to show improvements in accumulation of either eflornithine or nifurtimox with NECT or when the individual drugs were in combinations with the other anti-HAT drugs. Interestingly, the combination of eflornithine and pentamidine caused a decrease in eflornithine accumulation, implicating an unidentified pentamidine-sensitive transport system – possibly an adenosinesensitive influx transporter. The cellular influx transport mechanisms used by eflornithine has been suggested to be those used by CAA due to the structural similarity of eflornithine with the CAA ornithine and so this was studied. We revealed in the hCMEC/D3s that eflornithine had degrees of sensitivity to a variety of transport mechanisms, in which system y+ appears to be the principal influx mechanism. Similar anti-HAT drug combination therapy studies with nifurtimox were performed and also illustrated a significant interaction with pentamidine; although conversely to eflornithine we demonstrated an increase in nifurtimox accumulation as a result of nifurtimox-pentamidine combination. Previous in situ observations by our group suggested nifurtimox was a substrate for efflux transport systems at the BBB that are separate from P-gp and this too was investigated, identifying the well known drug efflux pump BCRP as the principal nifurtimox efflux transporter. With regards to exogenous molecules, we provide evidence of CAA influx mechanisms for the anti-HAT drug eflornithine and an efflux system for nifurtimox, principally involving BCRP. We also found that NECT and combination therapy of eflornithine or nifurtimox with the other anti-HAT drugs did not improve eflornithine or nifurtimox accumulation when compared to controls – except when pentamidine was combined with nifurtimox. This finding suggests that nifurtimox-pentamidine combination may improve efficacy of nifurtimox in the field. Collectively, these data further demonstrate of the suitability of the hCMEC/D3 cell line as a powerful tool for human in vitro BBB investigation across a range of study areas.

This thesis describes studies to investigate the regulation of guanylate cyclases pathways in blood vessels. Endogenous iS-nitrosothiols have been implicated in the regulation of soluble guanylate cyclase (sGC), by modulating nitric oxide (NO) bioactivity. To determine whether the biotransformation of S-nitrosothiols by isolated rat aorta is enzyme-dependent, stereoisomers of S-nitrosoglutathione (GSNO), S-nitrosocysteine (CYSNO) and 5-mtroso-N-acetyl-pemcillamine (SNAP) were synthesised and their decomposition and relaxant effects characterised. Decomposition of these S-nitrosothiol stereoisomers by Cu(I), Cu(II), Cu/Zn superoxide dismutase (Cu/Zn SOD) or rat aorta was equivalent (P > 0.05). Similarly, the vasorelaxant activity of iS-nitrosothiol stereoisomers was equipotent (P > 0.05). The selective Cu(I) chelator, bathocuproine disulfonic acid (BCS), blocked the decomposition of 5-nitrosothiol stereoisomers by Cu(I), Cu(II), Cu/Zn SOD and rat aorta (PO.05) and significantly inhibited their relaxant effects (P < 0.05). These studies suggest that in rat aorta, there are no stereospecific vasorelaxant effects of S-nifrosothiols, consistent with non- enzymatic release of NO. Biotransformation of S-nitrosothiols is, in part, dependent on Cu(I) ions. The sensitivity of sGC to NO and particulate guanylate cyclase (pGC) to atrial natriuretic peptide (ANP) regulates vasodilatation in response to these mediators. To determine the role of endogenous NO as a feedback regulator of sGC and pGC, rat aorta was incubated in vitro with bacterial lipopolysaccharide (LPS) to mimic many aspects of sepsis. LPS produced "high output" NO from inducible nitric oxide synthase (iNOS) and reduced the potency of the direct (SPER-NONOate, sodium nitroprusside, GSNO and BAY 58-2667) and indirect (acetylcholine and histamine) sGC activators. iNOS (1400W) but not cyclooxygenase (indomethacin) inhibition, preserved acetylcholine- and SPER-NONOate-dependent relaxations in LPS-treated vessels (PO.05). LPS reduced the potency of 8-bromo-cyclic guanosine-3',5'- monophosphate (PO.05), but not forskolin (adenylate cyclase activator) or 8-bromo- cyclic adenosine-3',5'-monophosphate (P > 0.05). LPS also reduced the potency of the pGC activators C-type natriuretic peptide and ANP. 1400W, the sGC inhibitor 1H- l,2,4 Oxadiazolo 4,3-a quinoxalin-l-one (ODQ) or both, preserved relaxations to ANP in LPS-treated vessels. 1400W and/or ODQ also reversed established desensitisation to ANP within minutes (all PO.05 versus LPS alone). These results indicate that sGC and pGC signalling are desensitised following exposure to LPS, consistent with a compensatory mechanism offsetting high-output NO production by iNOS. This effect is specific to cGMP-dependent pathways since the cAMP pathway was unaltered. The mechanism of desensitisation is likely to involve a reversible biochemical change, since the responsiveness was restored immediately following removal of excess NO (by 1400W) or cGMP (by ODQ). These observations suggest that inflammatory cardiovascular disorders associated with excessive NO production (i.e. septic shock) are characterised by specific impairment of GC-cGMP-mediated vasorelaxation that is mediated, at least in part by cGMP, and readily reversible. To investigate the mechanism(s) of iNOS-derived NO-mediated desensitisation of sGC and pGC, pharmacological inhibition of phosphodiesterases (type 5 (sildenafil), 1A1 (vinpocetine), and 3 (milrinone)), protein phosphatase 2A (okadaic acid and cantharidic acid), superoxide anion (SOD) and myeloperoxidase (4-aminobenzoic hydrazide) were used to probe the role of these enzymes. Desensitisation of ANP- and NO-mediated dilatation by iNOS-derived NO was unaffected by inhibition of these enzymes (all P > 0.05 versus LPS alone). However, activation of protein kinase C by phorbol 12-myristate 13-acetate and thymeleatoxin caused desensitisation of both guanylate and adenylate cyclases. If PKC activation causes LPS-induced desensitisation of sGC and pGC, then other mechanisms must preserved cAMP-mediated relaxation.

Electricity demand and renewables penetration are set to increase worldwide over the coming decades as part of the global decarbonisation effort. As a result, distribution networks are expected to face challenges related to increased peaks and undesirable voltage excursions. Hence, significant network reinforcements may be required over the next decades. However, a very significant challenge in realizing this transition is the increased uncertainty that surrounds future distributed generation and load connections in terms of size, location and timing. This uncertainty inadvertently will give rise to the prospect of inefficient investments and stranded assets given that current planning practices remain deterministic. It follows that new planning frameworks are needed that allow the quantification of option value and achieve reduction of stranding risk by encouraging cost-efficient strategic investments through smart technologies under both endogenous and exogenous sources of uncertainty. This thesis presents multi-epoch stochastic optimization models, for the distribution network planning problem, that consider a set of investment options with different techno-economical characteristics so as to reflect the multitude of choices available to planners in a realistic setting characterized by endogenous or exogenous uncertainty. These optimization models are rendered tractable through the use of novel decomposition schemes that effectively help manage the associated increased computational burden. The corresponding simulation results validate that smart technologies constitute valuable options for enabling cost effective integration of distributed generation units and underline the importance of early investment in such assets under decision-dependent uncertainty. In addition, the results emphasize that deterministic approaches systematically undervalue the flexibility that smart assets provide, thereby posing a barrier to the advent of the flexible smart grid paradigm.

Exercise has become a vital component of the therapy regimen prescribed to cystic fibrosis (CF) patients due to its systemic benefits, such as increased sputum expectoration, attenuation of the expected 2-3% annual decline in pulmonary function, and extended life expectancy. However, exercise still is not viewed as being as beneficial as pharmacological treatments by many CF patients and can be intimidating. My aims in this study were two-fold; first, to determine the ideal exercise intensity for individuals with CF; and second, to determine if exercise at this ideal intensity could provide improvements in ion regulation in the lungs, which was measured using exhaled breath condensate (EBC) collection and nasal potential difference (NPD), that were comparable to one of their standard pharmacological therapies, albuterol. I hypothesized that with moderate intensity exercise, Na⁺ absorption would decrease from baseline due to Na⁺ channel inhibition, rather than increase or remain unchanged, as was expected with albuterol, and cause an even greater increase Cl- secretion compared to albuterol due to activation of both CF-dependent and independent Cl- efflux with exercise. CF (n=14) and healthy (n=16) subjects completed three visits, a baseline screening and two treatment visits. I collected EBC at baseline, 30- and 60-minutes post-albuterol administration on one visit, and at baseline and during three separate 15 min exercise bouts at low, moderate and high intensity on the other visit. Following the EBC collection, NPD was performed at 30- and 80-minutes post albuterol or following moderate and high intensity exercise. We also measured spirometry and diffusing capacity of the lungs for nitric oxide (DLNO) during each visit at the various time points. In CF subjects, moderate intensity exercise resulted in greater improvements in DLNO (39 ± 29vs.15 ± 22% change from baseline, exercise vs. albuterol respectively), similar levels of bronchodilation compared to 60-minutes post-albuterol administration, no change in Na⁺ absorption, and a four-fold increase in Cl- secretion. Our results suggest that moderate intensity exercise is the best dose for CF patients, and can provide comparable changes as its pharmacological counterpart albuterol, when compared over a short term duration.

Type 2 diabetes mellitus results from a combination of insulin resistance and impaired insulin secretion. The aim of this study is to investigate the effect of endogenous and exogenous cannabinoids on insulin resistant cell lines, viz skeletal muscle (C2C12) and fat (3T3-L1), and to investigate the effects of these cannabinoids on insulin secretion in pancreatic β-cells (INS 1). Insulin resistance was induced in the cells using 20 ng/mL TNF-α (3T3-L1) and 100 nM insulin (C2C12). Insulin resistant cells were exposed to cannabinoids for 48 hours after which glucose uptake, RT-PCR and Western blot analysis was performed. Additionally, adipokine assays were performed on the 3T3-L1 cells. The insulin resistant 3T3-L1 and C2C12 cells had reduced glucose uptake, decreased IRS-1 and Glut-4 expression indicative of an insulin resistant state. The extract and THC significantly enhanced glucose uptake, IRS-1 and Glut-4 in 3T3-L1 and C2C12 cells. The extract and THC thus have the potential to be an insulin sensitizing agent. Interleukin-6 was significantly decreased by THC. INS 1 cells, cultured under normoglycemic conditions, were exposed to cannabinoids for 48 hours after which glucose-stimulated insulin secretion, radioimmunoassay, oxygen consumption, RT-PCR and Western blot analysis was performed. Insulin stimulatory index was not significantly affected after cannabinoid exposure, except by THC. The cannabinoids decreased insulin content, in a concentration dependent manner, but the inhibition mechanism remains elusive. The cannabinoid Treated cells showed insulin gene expression levels similar to the control, while only THC proved effective in significantly stimulating Glut-2 gene expression. Oxygen consumption studies showed levels lower than the control cells. Most of the cannabinoids inhibited insulin secretion under normoglycemia except THC, while the cannabinoids exhibited the potential to improve insulin resistant adipocyte and myocytes response to glucose and gene regulation.

CD23 is the low affinity IgE receptor. It is a type II integral transmembrane glycoprotein that can be shed from the cell surface forming soluble products of approximately 37, 33, 29, 25 and 16kDa. It appears that membrane and soluble CD23 have opposing regulatory functions and that inhibition of CD23 shedding may have potential to alleviate both allergic and inflammatory diseases. This has focused attention on the endogenous protease(s) responsible for CD23 shedding, leading to the demonstration that both the 37 and 33kDA sCD23 fragments are cleaved from the cell surface by a metalloprotease. In order to characterise the cleavage events within CD23, anti-neoepitope antibodies specific for the newly created amino and carboxy termini of the two predominant cleavage sties within CD23 (producing the 37 and 25kDa soluble CD23 products) were raised. Characterisation of these antibodies demonstrated that the proteolytic cleavage events responsible for creating the 37 and 25kDa sCD23 fragments are independent of each other. Furthermore, two different proteases were shown to be responsible for cleaving these two fragments. The work described in this thesis confirms previous reports that 37kDA sCD23 is cleaved by a metalloprotease, however cleavage of the 25kDa fragment was not inhibited by metalloprotease inhibitors. The production of the two different sized sCD23 molecules by different proteases has important implications for targeting the proteolytic cleavage events to alleviate symptoms of allergic and inflammatory diseases. This emphasises the importance of defining the biological functions of mCD23 and each of the sCD23 molecules.

The aim of this study was to examine apo(a)B particles distribution among plasma lipoproteins in two situations in which triglyceride-rich lipoproteins are increased: in normolipidemic subjects after a fat load and in patients with endogenous hypertriglyceridemia.
The results obtained demonstrate that, in hypertriglyceridemic patients, the distribution of apo(a)B particles among lipoprotein fractions is different than that observed in normotriglyceridemic subjects. These differences in distribution are a result of the association of apo(a)B particles with TG-rich lipoproteins. Contrary to apo(a)B particles found in the cholesterol-rich fraction apo(a)B particles in the TG-rich fraction are associated with lipoproteins of different sizes. The amount of apo(a)B particles in the TG-rich fraction could represent up to 50% of its total concentration and may contribute to the atherogenic and the thrombogenic potential of VLDL in HTG.

An investigation of the interaction of stable opioid/ligands and unstable opioid peptides with opioid receptors in guinea pig brain, guinea pig myenteric plexus and mouse vas deferens has been carried out. The initial aim of the study was to further characterise K opioid receptors, using binding assays and isolated tissue bioassays. The second aim was to determine the true affinity and potency of small dynorphin peptides for the K opioid receptor and to determine if metabolism of the peptides to non K opioid receptor-preferring products contributes to their observed in vitro pharmacology.

This study investigated the effect of endogenous and exogenous heating on body core cooling rate, afterdrop and re-warming rate in humans during subsequent cold-water immersion. On three separate days, following 30 min baseline resting at an ambient temperature of 25°C, seven subjects were either: (1) seated upright for 15 min rest (Control); (2) exercised 15 min on a cycle ergometer at 70% VO2 max (Exercise); or, (3) immersed 16.8 +/- 6.2 min in warm water (40°C) (Warm-water immersion) to an esophageal temperature (TES) similar to that of end of exercise. Subjects were then cooled in water (7°C) to a TES of 34.5°C and rewarmed by spontaneous shivering (shivering thermogenesis) in air. There was a 1.3-fold increase in the overall body core cooling rate during Exercise as compared to Control (P < 0.01). Warm-water immersion demonstrated the largest difference in overall cooling rate above Exercise and Control with a 2.5- and 3.3-fold increase, respectively (P < 0.01). The greatest difference between trials occurred within the initial 15 min (i.e., a 2.0- and 4.9-fold increase above Control for Exercise and Warm-water immersion, respectively). There was no significant difference in the TES afterdrop (∼0.5°C) nor afterdrop duration (∼15 min). Similarly, rewarming rates were almost identical under all conditions (∼3.1°C·h-1). These data show that pre-warming can have a detrimental effect on survival time during a subsequent cold-water immersion.

This dissertation addresses the modeling and solution of mixed-integer linear multistage stochastic programming problems involving both endogenous and exogenous uncertain parameters. We propose a composite scenario tree that captures both types of uncertainty, and we exploit its unique structure to derive new theoretical properties that can drastically reduce the number of non-anticipativity constraints (NACs). Since the reduced model is often still intractable, we discuss two special solution approaches. The first is a sequential scenario decomposition heuristic in which we sequentially solve endogenous MILP subproblems to determine the binary investment decisions, fix these decisions to satisfy the first-period and exogenous NACs, and then solve the resulting model to obtain a feasible solution. The second approach is Lagrangean decomposition. We present numerical results for a process network planning problem and an oilfield development planning problem. The results clearly demonstrate the efficiency of the special solution methods over solving the reduced model directly. To further generalize this work, we also propose a graph-theory algorithm for non-anticipativity constraint reduction in problems with arbitrary scenario sets. Finally, in a break from the rest of the thesis, we present the basics of stochastic programming for non-expert users.

Opioids are responsible for a number of effects but are employed primarily as analgesics. The discovery of endogenous opioids and multiple receptors have led to a better understanding of how analgesics function, and how both opioid receptors and endogenous ligands are regulated. The hypothesis of this dissertation states that levels of endogenous enkephalins are modulated by stress, inflammation and the endogenous peptide cholecystokinin (CCK) to alter the antinociceptive efficacy of μ and, possibly, δ opioids. Endogenous enkephalin release results in either direct antinociception or synergistically enhances the antinociceptive effects of the μ agonist morphine via δ receptors. This thesis will first detail how the administration of exogenous δ compounds can enhance the antinociceptive effects of a μ agonist. Exposure of mice to a cold-water swim-stress (CWSS) paradigm resulted in direct antinociception that was attenuated by opioid antagonists. Exposure to CWSS for a limited amount of time did not produce significant antinociception, but produced a marked enhancement of morphine antinociception. This enhancement was blocked by the administration of δ antagonists and (Leu⁵) enkephalin antisera. An antisense oligodeoxynucleotide was designed from the δ opioid receptor and administered to animals. Animals treated with the δ antisense and exposed to the CWSS paradigm showed a significant decrease in antinociception. Inflammation produced in the hind-paw of the mouse significantly enhanced the antinociceptive effects of morphine that was inhibited by δ antagonists and (Leu⁵) enkephalin antisera. The administration of a selective CCK(B) antagonist, L365,260 or CCK(B) antisense, enhances the antinociceptive potency of morphine. This enhancement is attenuated by δ antagonists, (Leu⁵) enkephalin antisera, as well as exhibits a two-way cross tolerance with δ agonists. L365,260 and an "enkephalinase" inhibitor, when coadministered, produced significant antinociception that was blocked by a δ antagonist and (Leu⁵) enkephalin antisera. Using polymerase chain reaction in search of a δ opioid receptor subtype, an orphan receptor was cloned and characterized as a member of the G protein-coupled receptor family. These data suggest that stress results in the release of (Leu⁵) enkephalin that enhances the antinociceptive effects of a μ-selective agonist morphine. Release of endogenous enkephalins may be regulated by the interactions of cholecystokinin at the CCK(B)receptor. Such information may lead to appropriate use of opiates in conjunction with CCK antagonists for the management of appropriate pain states.