Дисертації з теми "Mechanism of action of electroconvulsive therapy"

Background: Electroconvulsive therapy (ECT) is one of the most effective and fast acting therapeutic options for treatment-resistant psychiatric diseases, in particular mood disorders. Mood disorders are highly heterogeneous, disabling and severe mental illness, at still unknown etiology, which have been associated with a multifaceted pathogenesis, encompassing genetic, epigenetic and metabolic vulnerabilities together psychosocial/lifestyle factors. Among the various biological patterns thought to be involved in the physiopathology of dysthymia, major depression, cyclothymia, bipolar I, II, mixed states and related disorders, alterations in the neuroendocrine and immune systems have been also evidenced. Additionally, an increasing number of studies have highlighted the relevance of impaired mechanisms of defense against reactive oxygen species (ROS) in the progression and severity of BD. Oxidative stress and redox states are indeed part of the metabolic and chemical networks of the immune/inflammation response. Despite such evidences, few studies have examined, by now, the impact of ECT on specific neuroendocrine, immune and oxidative stress paths in patients undergoing this therapy. It has been reported that ECT-induced epileptic seizures stimulate the intra-cerebral release of cytokines, including the cytokine network associated with the pathophysiology of affective disorders. It is therefore challenging to consider that the therapeutic efficacy of ECT may reside on the degree of activation of the immune/inflammatory system and that patients, under depressive, hypomanic, manic or mixed states, may change their specific profile of biochemical/immunological markers by ECT. ECT would thus act on complex biochemical cascades, formed by several neurotransmitters, neuro-hormones, neurotrophic factors and metabolic substrates, playing a significant therapeutic role. Hypothesis: Beside possible neurotransmitter and neurotrophin variations, mood disorder patients would also present significant changes of peripheral cytokine and oxidative stress profiles, before and after ECT; it might be thus possible to identify specific redox chemical and immune features related to the response/Non-response to this treatment. Such a result could also considerably help to detect peripheral molecular correlates of immunochemical dysregulation, refractory symptoms and ECT therapeutic benefits or adverse effects in mood disorders. Aims: The foremost aims of this study were: 1) to explore the degree of expression/activity of peripheral immune and oxidative stress markers during the course of ECT in bipolar patients; 2) to possibly evidence differences of these biochemical parameters among Remitter and Non-Remitter patients, assessed by means of suitable examinations and psychometric questionnaires, administered to recruited patients before, during and after ECT. Methods: From 2016 to 2018, we recruited and investigated 17 consecutive patients with a BD diagnosis accordingly to DSM-V diagnostic criteria, all treated by ECT at the Psychiatry section of the Department of Clinical and Experimental Medicine of the University of Pisa. All patients were examined during three main phases of ECT course, following this schedule: 1) at T0, by both psychometric and biochemical evaluations, before the first application; 2) at T1, by biochemical evaluations carried out 3 hours after the first application; 3) at T2, at the end of the treatment, by both psychometric and biochemical examinations. A forth biochemical assessment was carried out also at T3, 3 hours after the last application, in order to possible appraise a different reactivity of immune/oxidative stress patterns at the end of ECT applications. To constantly monitor patients, psychiatric and physical examinations were always performed for the duration of the study. Psychometric questionnaires carried out prior to (T0) and after (T3) the ECT course, consisted into: the Hamilton Depression Rating Scale-17 (HAM-D), Young Mania Rating Scale (YMRS), Brief Psychiatric Rating Scale (BPRS) and Clinical Global Improvement Impression (CGI) scale. The CGI subscale “global improvement”, final HAM-D and YMRS total scores were used to identify Remitter and Non-Remitter groups . Biochemical investigations, performed after blood samplings carried out at the 4 scheduled times, T0, T1, T2 and T3, were: 1)-the plasma levels of the immune/inflammatory cytokines IL-6, IL-8 and TNFα; 2)-the plasma levels/ activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), total thiols (R-SH), the ferric reducing ability of plasma (FRAP), uric acid as well as an index of oxidative damage, the advanced oxidation plasma protein products (AOPP). Results: At the end of therapy, about the 53% of ECT-treated BD patients was found to remit. Concerning biochemical investigation, we observed that, globally, in 17 subjects, 3 hours after ECT (T1), the activity of SOD in plasma increased nearly attaining the statistical significance, while FRAP was found significantly decreased; when analyzing Remitters and Non-Remitters separately, the nearly significant increased SOD reported in all patients at T1, after the first ECT application, was due to a greater enzyme activity in Remitters, while the global T1 FRAP reduction was due to the significant decrease of plasma ferric reducing power in Non-Remitters only. We also reported that Non-Remitters had a significantly reduced CAT activity both at T1 and in the long term, at the end of ECT course (T2), and a higher percent of responce in uric acid and IL 8 after the last ECT (T3 vs. T2). Also, Non-Remitters tended to concomitantly have, at T2, higher plasma FRAP, SOD,IL6 and lower CAT, Thiols in respect to baseline (T0) values as well as in respect to Remitters. No significant effect on the plasma level of AOPP was observed at any scheduled time in all patients, indicating that no relevant protein damage, due to ROS was reported during ECT sessions. Limitations: The study, at the present stage, had a small sample size; moreover the patient group was heterogeneous, consisting of treatment resistant bipolar patients in different phases of illness and with different pharmacological regimes. Conclusions: According to literature, we showed that ECT is an effective and safe treatment able to heal drug-resistant bipolar patients with very severe clinical presentation and risk of suicide, in all phases of the illness. Preliminary results suggest that ECT can induce changes of the antioxidant system: an increased ROS scavenging activity at T1 seems to be an index of favorable response. The diminuition of antioxidant defense system would be conversely linked to reduced benefits deriving from this therapy. The recruitment of a larger cohort of patients is needed to confirm and pursue this useful investigation of peripheral biomarkers of ECT response. This will permit to perform more robust statistical tests as multivariate regression or principal component analyses and to possibly define peculiar immunochemical changes related to the clinical response.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007.
Vita. Briscoe Library received only one copy of this dissertation. It is shelved in the Archives for safekeeping. Includes bibliographical references.

Les stratégies de traitement biologiques ciblant le TNF-α se sont avérées efficaces pour réduire l'inflammation et les symptômes cliniques dans plusieurs maladies inflammatoires chroniques et sont maintenant couramment utilisées pour les patients qui ne répondent pas aux AINS au cours de la spondyloarthrite (SpA). Cependant, 30 à 40% des patients ne répondent pas aux anti-TNF, et il est actuellement impossible de prédire la réponse des patients à ces biomédicaments. Pour améliorer les résultats cliniques, nous avons besoin d’une part d’une meilleure compréhension des mécanismes d’action des anti-TNF sur le système immunitaire, et d’autre part de biomarqueurs permettant de prédire la réponse à ces biomédicaments afin de guider la décision thérapeutique. Mon projet de doctorat a porté sur deux objectifs complémentaires: (i) l'objectif principal était de progresser dans notre compréhension des mécanismes pathogéniques impliqués dans la SpA axiale et de définir de quelle façon les anti-TNF-α affectent les réponses immunitaires des patients, (ii) de développer des biomarqueurs pour prédire la réponse thérapeutique aux inhibiteurs du TNF. En collaboration avec l'équipe du Pr. Dougados à l'Hôpital Cochin, nous avons recruté deux cohortes indépendantes de patients SpA ayant une maladie active et pour lesquels nous avons collecté des échantillons de sang avant l'initiation du traitement par anti-TNF puis 1 semaine et 3 mois après le début du traitement. Les réponses immunitaires de ces patients ont été analysées à l'aide de tests hautement standardisés réalisés ex-vivo sur sang circulant. Ces tests "TruCulture" se présentent sous forme de seringues, dans lesquelles 1 ml de sang total est mis à incuber avec un stimulus spécifique ; 20 stimuli différents ont été testé et validé avant et après traitement dans les deux cohortes de patients. Nous avons observé une réduction très significative de la sécrétion de IL-1ra, IL-1β, IL-8, and MIP-1β en réponse à des stimuli microbiens et à des agonistes des TLR dans les échantillons de sang prélevés 7 jours et/ou 3 mois après le début du traitement. Pour identifier les bases moléculaires de l’action des inhibiteurs du TNF nous avons analysé l'expression des gènes dans ces différentes conditions de stimulation. L'analyse bioinformatique quantitative de l'expression des gènes (QuSAGE) a révélé que les gènes les plus modulés par le traitement anti-TNF étaient NF-KB et les gènes cibles de NF-kB, y compris le TNF lui-même et l’IL1B. Nos données suggèrent que les inhibiteurs du TNF agissent principalement en perturbant une boucle autorégulatrice pilotée par NF-kB. Afin d'identifier les signatures immunologiques de réponse aux anti-TNF avant le début du traitement, nous avons corrélé les réponses immunitaires chez les patients analysés au temps 0 à la réponse thérapeutique aux anti-TNF mesurée à 3 mois. Nos résultats suggèrent que les patients atteints de SpA et exprimant des niveaux inférieurs de PAX5 et des niveaux supérieurs de SPP1 en réponse à la stimulation avec SEB avant l'initiation de la thérapie anti-TNF ont les meilleures réponses thérapeutiques. Notre recherche montre que les tests TruCulture sont un outil efficace pour étudier les fonctions immunitaires chez les patients atteints de SpA et que les effets du traitement anti-TNF peuvent être mesurés lorsque les cellules immunitaires sont stimulées. En terme de recherche translationnelle, nous avons identifié des molécules qui pourront être utilisés comme biomarqueurs pour aider les cliniciens à prédire les réponses thérapeutiques aux traitements anti TNF
The introduction of anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in several chronic inflammatory diseases. However, 30-40% of patients do not respond to TNF blockers and it is currently not possible to predict responsiveness of patients to anti-TNF therapy. Furthermore, their impact on the immune system is incompletely understood. The goals of my PhD project were (i) to define the impact of anti-TNF therapy on immune responses to microbial challenges and stimuli targeting specific immune pathways in spondyloarthritis (SpA) patients, and (ii) to identify immunological correlates associated with therapeutic responses to TNF-blockers.Using a set of whole-blood, syringe-based assays to perform ex vivo stimulation while preserving physiological cellular interactions (TruCulture assays), we have performed a pilot study in SpA patients and investigated immune responses to 20 different stimuli before and 3 months after initiation of anti-TNF therapy. These findings were validated in a replication cohort, also assessing the effects of anti-TNF agents after only one week of treatment. We observed a highly significant reduction of the secretion of IL-1ra, IL-1β, IL-8 and MIP-1β in response to selected stimuli after 3 months of treatment compared to the baseline. Interestingly, these changes were already detectable after a single injection of an anti-TNF agent. To gain insight into the molecular mechanism of TNF blockers, we profiled gene expression in the stimulation cultures from all patients. Quantitative set analysis for gene expression (QuSAGE) revealed that the gene modules most affected by anti-TNF therapy are NF-kB transcription factors and inhibitors and NF-kB target genes, including TNF itself and IL1B. Our data suggest that TNF-blockers primarily act by disrupting an autoregulatory loop driven by NF-kB. We also tested whether there is a correlation between the responses of immune cells to specific stimuli and the clinical response to TNF-blockers. The decision tree model that we trained and validated suggests that SpA patients who expressed lower levels of PAX5 and higher levels of SPP1 in response to SEB stimulation before initiation of anti-TNF therapy had the best therapeutic responses. Our study shows that TruCulture assays are an efficient and robust tool to monitor immune functions in SpA patients and that the effects of anti-TNF therapy can be measured when immune cells are challenged, but not at steady state. Our data also indicate that analyzing immune responses in patients before therapy is a promising strategy to develop biomarkers for prediction of therapeutic responses to TNF-blockers

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives (norethisterone enanthate (NET-EN); norethisterone acetate (NETA)), are used by millions of women as contraceptives and in hormone replacement therapy (HRT). Although both progestins are widely used, very little is known about their mechanism of action at the molecular level. In this thesis, the differential regulation of gene expression and molecular mechanism of action via different steroid receptors by these synthetic progestons, as compared to progesterone (Prog) was investigated in human cell lines. In the first part of the study, the effect of Prog, MPA and NET-A on the expression of endogenous cytokine genes was investigated in two epithelial cell lines of the human female genital tract, Ect1/E6E7 (an ectocervical cell line) and Vk2/E6E7 (a vaginal cell line). Quantitative realtime RT-PCR (QPCR) showed ligand-specific and cell-specific regulation of the interleukin (IL)-6, IL-8 and RANTES (Regulated-upon-Activation, Normal T cell Expressed and Secreted) genes with Prog, MPA and NET-A. Moreover, the repression of the TNF -induced RANTES gene by MPA in the Ect1/E6E7 cell line was found to be mediated by the androgen receptor (AR). The second part of the study focused on elucidating the androgenic activities of these two progestins, in comparison to Prog. Competitive binding in whole cells revealed that Prog, MPA and NET-A have a similar binding affinity for the hAR as the natural androgen dihydrotestosterone (DHT). Both transactivation and transrepression transcriptional assays demonstrate that, unlike Prog, MPA and NET-A are efficacious AR agonists, with activities comparable to DHT. Using a mammalian two-hydrid assay, it was shown that MPA and NET-A exert their androgenic actions by different mechanisms. NET-A, like DHT and other well-characterised androgens, induces the ligand-dependent interaction between the NH2- and COOH-terminal domains (N/C-interaction) of the AR independent of promoter-context, while MPA does this in a promoterdependent manner. In the third part of this study, competitive binding revealed that MPA and NET-A have a similar binding affinity to each other, but about a 100-fold lower affinity than Prog for the human mineralocorticoid receptor (hMR), while RU486 has an even lower affinity for the hMR. Promoter-reporter assays showed that MPA, NET-A and RU486 are all antagonists of the hMR, but unlike Prog, they have weak antagonistic activity. However, on the endogenous MR-regulated Orm-1 (a-glycolytic protein or orosomucoid-1) gene expressed in a rat cardiomyocyte cell line, NET-A and RU486, but not MPA, has similar antagonistic activity as Prog. This study is the first to show that, NET-A and RU486, but not MPA, can dissociate between transrepression and transactivation via the hMR. Taken together, these results show that natural Prog and the synthetic progestins, MPA and NET-A display differential promoter-, cell- and receptor-specific effects on gene expression. Furthermore they may have important implications for cervicovaginal immune function, cardiovascular and other physiological functions.
AFRIKAANSE OPSOMMING: Medroksieprogesteroon asetaat (MPA), noretisteroon (NET) en derivate daarvan (noretisteroon enantaat (NET-EN); noretisteroon asetaat (NET-A), word deur miljoene vroue gebruik as voorbehoedmiddels en vir hormoon vervangingsterapie (HVT). Tenspyte daarvan dat beide hierdie progestiene algemeen gebruik word, is min bekend oor hulle meganisme van werking op molekulêre vlak. In hierdie proefskrif word die differensiële regulering van geenuitdrukking asook die molekulêre meganisme van werking deur middel van steroïedreseptore van beide hierdie sintetiese progestiene, ondersoek, en vergelyk met progesteroon (Prog), in menslike sellyne. In die eerste deel van die studie is die effek van Prog, MPA en NET-A op die uitdrukking van endogene sitokinien gene ondersoek in twee epiteel sellyne van die menslike vroulike geslagskanaal, Ect1/E6E7 (‘n ektoservikale sellyn) en Vk2/E6E7 (‘n vaginale sellyn). Kwantitatiewe intydse RT-PKR het ligand-spesifieke en selspesifieke regulering van interleukien (IL)-6, IL-8 en RANTES (Regulering-na- Aktivering, Normale T-sel Uitgedrukte en Afgeskei) gene getoon met Prog, MPA en NET-A. Verder is gevind dat die onderdrukking van die TNF- - geïnduseerde RANTES geen deur MPA in die Ect1/E6E7 sellyn bemiddel word deur die androgeen reseptor (AR). Die tweede deel van die studie het gefokus op die toeligting van die androgeniese aktiwiteit van die twee progestiene in vergelyking met Prog. Kompeterende binding in volselle het getoon dat Prog, MPA en NET-A ‘n soortelyke bindings affiniteit vir die menslike AR as die natuurlike androgeen dehidrotestosteroon (DHT) vir die menslike AR het. Beide transaktiverings en transonderdrukkings transkripsionele analieses toon dat, anders as Prog, MPA en NET-A effektiewe AR agoniste is met aktiwiteite wat vergelykbaar is met die van DHT. Deur die gebruik van ‘n soogdier twee-hibried toets, kon gewys word dat MPA en NET-A hul androgeniese effekte uitoefen deur verskillende meganismes. NET-A, soos DHT en ander goed gekarakteriseerde androgene, induseer die ligand-afhanklike interaksie tussen die NH2- en COOH-terminale domeine (N/C-interaksie) van die AR, onafhanklik van die promoter-konteks. MPA, aan die ander kant, doen dit op ‘n promoter-afhanklike manier. In die derde deel van die studie het kompeterende binding getoon dat MPA en NETA soortelyke relatiewe bindings affiniteite vir die menslike mineralokortikoïed reseptor (hMR) het, maar dat hierdie affiniteit ongeveer 100-voud laer is as die van Prog en dat die affiniteit van RU486 vir hMR selfs nog laer is. Promoter-rapporteerder toetse het getoon dat MPA, NET-A en RU486 almal antagoniste van die hMR is, maar anders as Prog, is hierdie ‘n swak antagonistiese aktiwiteit. Nietemin, op die endogene MR-gereguleerde Orm-1 ( -glikolitiese proteïen of orosomukoïed-1) geen, uitgedruk in ‘n rot kardiomiosiet sellyn, het NET-A en RU486, maar nie MPA nie, ‘n soortgelyke antagonistiese aktiwiteit as Prog. Hierdie studie is die eerste om te wys dat NET-A en RU486, maar nie MPA nie, kan onderskei tussen transrepressie en transaktivering deur middel van die hMR. Samevattend toon die resultate dat natuurlike Prog en die sintetiese progestiene, MPA en NET-A, ‘n differentiële promoter-, sel- en reseptor-spesifieke effek op geenuitdrukking het. Verder mag die resultate belangrike implikasies vir servikovaginale immuunfunksie, asook kardiovaskulêre en ander fisiologiese funksies, inhou.

La voie de signalisation mTOR intègre une variété de signaux environnementaux pour réguler la croissance et le métabolisme cellulaire. Cette voie est altérée dans 70% des cancers. Les inhibiteurs allostériques de mTOR, comme la rapamycine et ses dérivés (évérolimus et temsirolimus), sont administrés aux patients atteints de tumeurs métastatiques du sein, du rein et neuroendocrines. Cependant, leur efficacité reste modeste et une majorité de patients rechutent. L'utilisation de rapalogues fait donc face à deux problèmes cliniques majeurs : 1/l’absence de biomarqueur qui permette de stratifier les patients qui bénéficieraient le plus d'un traitement par rapalogues ; 2/ l’existence de plusieurs mécanismes de résistance décrits ou non. L’objectif de mon travail de thèse est d’identifier des nouveaux gènes cible des rapalogues utilisables comme biomarqueurs prédicteurs de l’efficacité du traitement ou comme cibles thérapeutiques pour vaincre la résistance.Nous avons identifié le gène TRIB3 comme cible des rapalogues. Sous traitement, son expression est diminuée dans un panel de lignées tumorales et des échantillons tumoraux. Nous avons démontré que cette régulation est indépendante de l’inhibition de la voie mTOR, mais médiée par le répresseur transcriptionnel GCF2. Des analyses protéomiques à haut débit ont identifié TRIB3 en tant que composant du spliceosome. De plus, nous avons démontré que la régulation négative de TRIB3 est nécessaire aux rapalogues pour modifier l'épissage des pré-ARNm. A l’inverse, la surexpression de TRIB3 supprime ces effets des rapalogues. En conclusion, ce travail de thèse ouvre plusieurs perspectives: 1 / l'utilisation potentielle de TRIB3 comme biomarqueur pour prédire ou évaluer l'efficacité du traitement par les rapalogues; 2 / de nouvelles opportunités thérapeutiques ciblant ces mécanismes indépendants de mTor ; 3/ la combinaison possible des rapalogues avec des composés ciblant l’épissage afin de surmonter la résistance
The mTOR signaling pathway senses variety of environmental cues and integrates them to regulate cellular growth and metabolism. This pathway is altered in 70% of cancers. Allosteric inhibitors of mTOR like rapamycin and its derivatives (everolimus and temsirolimus) have become standard of care in patients with metastatic breast, kidney and neuroendocrine tumors. Unfortunately, their role is modest and most of patients will relapse. Thus, in clinic there are two major concerns related to the use of rapalogs: 1/ the absence of accurate biomarker to stratify patients who would benefit from rapalogs treatment; 2/ the existence of known and unknown mechanisms of resistance. Accordingly, the aim of my PhD project is to identify new target genes of rapalogs that could be used as biomarkers to predict treatment efficacy, or as therapeutic targets, to overcome resistance.We identified TRIB3 gene as a novel target of rapalogs. Upon treatment, its expression is down-regulated both in a panel of cancer cell lines and in cancer patient samples. We showed that this regulation is independent of the mTOR signaling inhibition, but relies on a transcriptional regulation via the co-repressor GCF2. High-throughput proteomic analyses identified TRIB3 as a component of the spliceosome. Additionally, we demonstrated that the down-regulation of TRIB3 is necessary for rapalogs to alter pre-mRNA splicing. In contrast, the, overexpression of TRIB3 abolishes these effects of rapalogs. In conclusion, this PhD work leads to the following important perspectives: 1/ the potential use of TRIB3 as a biomarker to predict or asses the efficacy of rapalogs treatment; 2/ new window of therapeutic possibilities by targeting this mTOR - independent mechanism of action; 3/ the potential combination of rapalogs with splicing targeting agents to overcome resistance

Bcr-abl expression being associated with anti-apoptotic signaling and expression of DNA repair enzymes, we surmised that single molecules capable of blocking abl tyrosine kinase (TK) function and damaging DNA should lead to compounds with potency superior to that of GleevecRTM. To this end, we designed novel agents termed "combi-molecules" programmed to not only behave as bcr-abl inhibitors on their own, but also to further degrade to another inhibitor and a DNA damaging species. The released inhibitor was designed to sustain bcr-abl inhibition following degradation of the combi-molecule and the DNA damaging species to activate pathways leading to apoptosis. To model this strategy termed "combi-targeting", we synthesized ZRCM5 (a monoalkyltriazene) that showed antiproliferative activity superior to that of the classical DNA damaging agent TemodalRTM, but not to that of Gleevec RTM. This result was imputed to the rather weak bcr-abl inhibitory activity of ZRCM5 and its strong DNA damaging property. Another prototype designed to contain an aniline mustard moiety (AK04) was a strong bcr-abl inhibitor but a poor DNA alkylating agent. Its cytotoxic activity was again stronger than that of the clinical alkylating agent chlorambucil but inferior to that of GleevecRTM. Further chemical studies directed at structural modification of the benzamide moiety led to the synthesis of ZRF1 with strong potency against bcr-abl TK and strong DNA damaging property. This novel optimized combi-molecule showed a 1.6-3-fold greater potency than GleevecRTM against bcr-abl expressing cells. Further investigation with ZRF1, showed that its cytotoxic potency was dependent on the p53 wild-type status of the cells. In cells expressing wild-type p53, p21 transactivation was associated with cell cycle arrest and that of Bax with apoptosis. In addition to, the pro-apoptotic effect of bcr-abl inhibition, these multiple mechanisms of action may synergistically enhance the cytotoxic potency of ZRF1 in p53 wild-type cells. The study conclusively demonstrated that p53 is a major determinant for the cytotoxic advantage of the novel combi-molecular approach in chronic myelogenous leukemia (CML), a disease in which 70-85% of all cases express wild-type p53.

- Introducció Alzheimer's disease (AD) is a neurodegenerative disorder characterized by early synaptic and late neuronal loss, affecting more than 26 million people worldwide. Among patients affected with dementia, more than half suffer from Alzheimer’s disease. The biggest risk factor for developing Alzheimer's disease is age. β-amyloid (Aβ) plaques and neurofibrillary p-Tau tangles accumulates in the brains of elderly patients playing a central role in the pathogenesis of AD. During the last years the fruit fly, Drosophila melanogaster has increasingly been used as a model for neurodegenerative disease. Although the adult fly has a simpler nervous system than those of vertebrates, it is capable of higher-order brain functions, including aversive and appetitive learning, and recalling learned information from prior experiences. - Contingut de la investigació This work has been focused on modeling Alzheimer's Disease in Drosophila by expressing two human genes associated with AD (Aβ42 and Tau) in the fly central nervous system. This model displays AD-like neuropathological as well as behavioral symptoms. The main goal of developing such a model is to analyse and study the effect of new synthetic fatty acids molecules in the pathogenesis of AD. Additionally, the model organisms established in this study could provide tools that help to understand disease-specific processes resulting in neuronal loss. This study argues that Drosophila can be used to study the behavioural basis of human neurodegenerative diseases and may provide a model to identify novel therapeutic avenues for neurodegenerative diseases as Alzheimer’s disease. In this work also was studied the effect of membrane lipid therapy on cognitive decline of a transgenic model of Drosophila. This model overexpresses the human amyloid peptide of 42 amino acids (Aβ42), and human Tau protein that play a key role in the development of this disease. - Conclusió The treatment has been based on the use of DHA and its hydroxylated derivate OHDHA, ARA and its hydroxylated form OHARA and EPA and its hydroxylated form OHEPA at 1, 3, 10, 30, 100 and 250 μg/ml of standard food. After testing the transgenes expression in the F1 generation by PCR analysis and Western blot it was evaluated the toxicity of the compounds, and it was demonstrated that food supplementation with OHDHA, OHARA, OHEPA partially restored the loss of locomotor activity and increased the life-span of the flies expressing the human transgenes whereas the DHA, ARA, EPA, form had not significant effects. It has been observed that the concentrations of 30 and 100 μg/ml of hydroxylated form, including the mixtures of (OHDHA+OHARA), (OHEPA+OHARA), and 30 μg/ml of TGMs, LP183A1, LP183A2, was used, have led to cognitive improvement and have maintained or increased the lifespan with respect to the control group. In addition it was analyzed the lipid content from Drosophila heads by using gas chromatography and it was found that the food supplementation with either hydroxylated or non-hydroxylated compounds induced changes in the fatty acid profile of Drosophila. Furthermore it was discovered that the amount of short chain fatty acids (SCFA), from the heads of F1 treated with ARA, EPA and DHA was less than that from untreated F1 flies. Concerning the hydroxylated fatty acids, the reduction in the levels of short chain fatty acid (SCFA) was similar to that of the non-hydroxylated fatty acids. All food supplement tested induced an increase of long chain fatty acids (≥ 18C). ARA, EPA and DHA were present in the fatty acid profile of flies treated with the respective non-hydroxylated food supplements. This fact proves the absorption and incorporation of dietary PUFAs into the Drosophila body tissues.
- Introducció La enfermedad de Alzheimer (AD, del inglés Alzheimer's disease) es una patología neurodegenerativa caracterizada por una pérdida temprana de conexiones sinápticas y, de manera tardía, de neuronas. Esta enfermedad afecta a unos 40 millones de personas en todo el mundo. Entre las personas con demencia, más de la mitad sufren AD. El mayor riesgo para desarrollar la enfermedad de Alzheimer es la edad. De hecho, las placas β-amiloide (Aβ) y ovillos neurofibrilares de fosfo-Tau se acumulan en los cerebros de pacientes ancianos, jugando un papel central en la patogénesis de AD. Además, se han encontrado reducciones significativas en los niveles de los lípidos fosfatidiletanolamina y ácido docosahexaenoico (DHA) en el cerebro de pacientes con AD. Durante la última década, la mosca de la fruta (Drosophila melanogaster) se ha utilizado como modelo para enfermedades neurodegenerativas, debido a que puede ser utilizada para el análisis de conductas como el aprendizaje aversivo y apetitivo, así como su capacidad de utilizar la información aprendida de previas experiencias, aunque la mosca adulta presenta un sistema nervioso mucho más simple que el de vertebrados. - Contingut de la investigació La presente investigación se centra en la utilización de Drosophila como modelo de AD mediante la sobreexpresión de los genes humanos asociados con AD (Aβ42 y Tau) en el sistema nervioso central de la mosca. El principal objetivo de desarrollar este modelo es analizar y estudiar el efecto de ácidos grasos sintéticos novedosos en la terapia de la AD. Conjuntamente, los organismos modelo establecidos en este trabajo pueden constituir un sistema que permita la comprensión de los procesos específicos de la enfermedad que desencadena la pérdida neuronal. Con todo ello, el presente trabajo demuestra que se puede usar Drosophila para estudiar las bases comportamentales de las enfermedades humanas neurodegenerativas y puede suponer un modelo para identificar nuevas terapias para dichas enfermedades, tales como AD. Además, se ha estudiado el efecto de la terapia lipídica de membrana en el declive cognitivo del modelo transgénico de AD de Drosophila. - Conclusió Los tratamientos empleados se basan en el uso de DHA y su derivado hidroxilado OHDHA, ARA y su forma hidroxilada OHARA y EPA y su forma hidroxilada OHEPA, así como derivados de triacilgliceroles (triacilglicerol miméticos, TGM) a dosis crecientes y añadidos en la comida. Tras confirmar la expresión de los transgenes en la generación F1 de las moscas por PCR y western blot, se analizó la toxicidad de los distintos compuestos y se demostró que la suplementación de comida con OHDHA, OHARA, OHEPA restauró la pérdida de actividad locomotora, parcialmente, además, aumentó la vida media de las moscas expresando los transgenes humanos, mientras que DHA, ARA, EPA no presentaron efectos significativos. Se observó que las concentraciones de 30 y 100 μg/ml de las formas hidroxiladas, incluyendo las mezclas de (OHDHA+OHARA), (OHEPA+OHARA) y 30 μg/ml de TGMs, LP183A1, LP183A2, mejoraron la capacidad cognitiva y aumentaron la vida media con respecto al grupo control no tratado. También se analizó el contenido lipídico en membranas de la cabeza de moscas mediante cromatografía de gases y se observó que la suplementación de la comida, tanto con los compuestos hidroxilados como los no-hidroxilados estudiados, indujo cambios en el perfil de ácidos grasos de Drosophila melanogaster. Entre ellos, se observó una menor cantidad de ácidos grasos de cadena corta en cabezas de moscas F1 tratadas con ARA, EPA and DHA en comparación con moscas no tratadas. En cuanto a los ácidos grasos hidroxilados, presentaron un nivel similar en la reducción de los niveles de ácidos grasos de cadena corta. Además, todos los suplementos añadidos a la comida indujeron un aumento de los ácidos grasos de cadena larga (≥ 18C). Finalmente, se observó la presencia de ARA, EPA y DHA en el perfil de ácidos grasos de las moscas tratadas con el correspondiente ácido graso no-hidroxilado. Este hecho prueba la absorción e incorporación de los ácidos grasos poliinsaturados presentes en la dieta en los tejidos de la Drosophila.

Les infections et l’activation du système immunitaire stimulent l’hématopoïèse. L’activation des récepteurs Toll-like (TLRs) des cellules souches hématopoïétiques, par leur reconnaissance de motifs moléculaires portés par des agents infectieux, en oriente la différenciation vers les voies myéloïdes, renforçant la capacité de notre organisme à lutter contre les infections. Ici, nous avons étudié si les agonistes TLRs peuvent, au contraire, induire au sein de la moelle osseuse l’émergence de progéniteurs hématopoïétiques présentant des propriétés immunorégulatrices. Nous montrons que l’incubation de moelle osseuse de souris en présence de l’agoniste TRL-9, CpG-B, entraîne l’émergence d’une population de progéniteurs au stade pro-B (appelée CpG-proBs). Le transfert adoptif de seulement 60,000 CpG-proBs par receveur, à l’apparition des premiers signes cliniques, confère une protection à long terme dans deux modèles expérimentaux de maladies auto-immunes, le Diabète de Type I (T1D) et l’Encéphalomyélite Auto-immune Expérimentale (EAE). La migration, la différenciation, et les mécanismes cellulaires et moléculaires de cette population protectrice sont décrits et comparés entre ces deux modèles. Dans les deux modèles, les CpG-proBs migrent vers le tissu cible de la réponse auto-immune et se différencient en cellules B matures régulatrices. Dans le T1D, l’interféron-γ (IFN-γ) produit par les cellules T s’avère essentiel pour induire la surexpression de FasL à la surface des CpG-proBs, entraînant l’apoptose des cellules T effectrices. De plus, l’IFN-γ produit par les CpG-proBs réduit la production par les cellules T de l’IL-21, une cytokine pathogène majeure dans le T1D. La descendance des CpG-proBs est composée de précurseurs transitionnels B, de cellules B de la zone marginale et de cellules B folliculaires, exprimant de forts niveaux de FasL et toujours capables d’induire l’apoptose des cellules T, prolongeant ainsi le contrôle des cellules effectrices T auto-immunes in vivo. Dans l’EAE, l’IFNγ est indirectement responsable de la rétention des cellules T, par l’internalisation de CCR7, au sein des ganglions lymphatiques, inhibant ainsi leur migration au système nerveux central (SNC). Dans la moelle épinière, tissu cible de l’EAE, les CpG-proBs se différencient en cellules B220+CD5+CD1dhiCD11b+, secrétant la cytokine anti-inflammatoire IL-10. Enfin, la mobilisation des progéniteurs hématopoïétiques par un cocktail de facteurs hématopoïétiques confère à une sous-population multipotente au stade MPP2 la propriété d’augmenter l’expansion des Foxp3+ Tregs et de prévenir la survenue du diabète de type 1. Nous montrons que les MPP2 mobilisés s’avèrent également capables d’exercer un effet protecteur envers l’EAE. Leur capacité à induire l’expansion de Treg Foxp3+ au sein du SNC et à la périphérie joue un rôle essentiel dans la protection des souris envers l’EAE, puisque la déplétion des Treg abolit la protection déjà établie. Pour conclure, nous avons mis en évidence que diverses stimulations de l’hématopoïèse induisent l’émergence de nouvelles populations de progéniteurs hématopoïétiques qui présentent des propriétés immunorégulatrices et constituent de nouveaux outils de thérapie cellulaire des maladies auto-immunes
It is well known today that various infectious events or other stimuli of the immune system can trigger hematopoiesis. The hematopoeitic stem and/or progenitor cells express on their cell surface Toll-like receptors which can recognize molecular motifs of infectious agents. The stimulation of TLRs on hematopoietic stem cells favors their differentiation into myeloid lineages, reinforcing the capacity of our body to fight against the pathogens. Herein, we have investigated whether the stimulation of TLRs can induce, instead, the emergence within the bone marrow of selective progenitor cells with immunoregulatory properties. We show that incubation of bone marrow cells with the TLR-9 ligand CpG-B can induce a pro-B cell population (named CpG-proBs) whose adoptive transfer at low numbers of 60,000 cells provided long-lasting protection in two models of autoimmune diseases, Type I Diabetes (TID) and Experimental Autoimmune Encephalomyelitis (EAE) at the onset of clinical signs. The migration, differentiation and molecular mechanism of action of this protective population is described and compared between these two models. In both models, the CpG-proBs migrate to the target tissue of autoimmune responses and differentiate into more mature regulatory B cells. In TID, IFN-γ produced by both T and CpG-proB cells is essential for the upregulation of FasL at the surface of CpG-proBs, inducing the apoptosis of the effector T cells. In addition, IFN-γ reduced the T-cell production of IL-21, a major pathogenic cytokine in TID. The progeny of the adoptively transferred CpG-proBs, including transitional precursors B cells, marginal zone and follicular B cells, display high expression of FasL, promote apoptosis of effector T cells and prolong the control of autoimmune effector T cells in vivo. In EAE, IFN-γ was responsible for the restriction of T cells to the lymph nodes, inhibiting their homing to the CNS. IFN-γ indirectly induced the internalization of CCR7, a receptor required for the migration across the blood-brain barrier. In the spinal cord (target tissue in EAE), CpG-proBs differentiated into B220+CD5+CD1dhiCD11b+ cells secreting the anti-inflammatory cytokine IL-10. Finally, hematopoietic progenitor populations mobilized to the periphery by a cocktail of G-CSF and Flt3l, at the stage of MPP2, have already been shown to protect against TID by expanding the Foxp3+ Tregs. We evaluated them in the EAE model, showing that the ability of these mobilized progenitor cells to trigger the expansion of Foxp3+ Treg within the CNS and the periphery was necessary for providing protection to EAE mice since Treg depletion abrogated the protection once established. In conclusion, we provide evidence for the emergence of new populations of hematopoietic progenitor cells which can display immunoregulatory properties and might be used for cell therapy of autoimmune diseases

Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment for graft-versus-host disease (GvHD), the major barrier to successful allogeneic haematopoietic stem cell transplantation. Importantly, ECP results in immunomodulation without affecting relapse or infection rates. The mechanisms of action of ECP described so far include Iymphocyte apoptosis, cytokine modulation and induction of T regulatory cells (Treg). This study attempted to analyse direct and indirect effects of ECP on dendritic cells (DC) using an in vitro model (in vitro PUVA). DC underwent apoptosis after PUVA treatment, which was preceded by partial maturation of DC whereas stimulation through CD40 ligation was abrogated and IL-12 secretion was abolished. PUVA-treated DC skewed naive T cells towards a Th2 response. Employing the human skin explant model as a GvHD model, it was shown that modulation of immature DCs by in vitro PUVA-treated Iymphocytes downregulated GvH reactions. Further experiments demonstrated that activated T cells were more prone to undergo apoptosis after in vitro PUVA treatment. In the last section, blood samples sequentially taken from patients treated with ECP within a Phase II clinical trial in Newcastle were analysed by flow cytometry. Total and relative numbers of Treg increased independently of the response status. Likewise, the number of CCR4-positive CD4 (Th2) cells rose. In conclusion, this study describes direct and indirect effects of ECP/PUVA- treatment on DC and reveals that DC modulation may be an important factor for the altered immune response observed after ECP-treatment. Predominant killing of activated T cells might serve as an explanation of why there is no generalised immunosuppression. The finding of Treg and Th2 cell induction in the course of ECP-treatment generally goes in line with published literature and puts further emphasis on ECP's regulatory impact on the immune response.

Cognitive behavioral therapy (CBT) is efficacious for panic disorder with agoraphobia (PD/A). Nevertheless, the active ingredients of treatment and the mechanisms through which CBT achieves its effects remain largely unknown. The mechanisms of action in CBT (MAC) study was established to investigate these questions in 369 patients diagnosed with PD/A. The MAC study utilized a multi-center, randomized controlled design, with two active treatment conditions in which the administration of exposure was varied, and a wait-list control group. The special feature of MAC is the way in which imbedded experimental, psychophysiological, and neurobiological paradigms were included to elucidate therapeutic and psychopathological processes. This paper describes the aims and goals of the MAC study and the methods utilized to achieve them. All aspects of the research design (e.g., assessments, treatment, experimental procedures) were implemented so as to facilitate the detection of active therapeutic components, and the mediators and moderators of therapeutic change. To this end, clinical, behavioral, physiological, experimental, and genetic data were collected and will be integrated.

Cognitive behavioral therapy (CBT) is efficacious for panic disorder with agoraphobia (PD/A). Nevertheless, the active ingredients of treatment and the mechanisms through which CBT achieves its effects remain largely unknown. The mechanisms of action in CBT (MAC) study was established to investigate these questions in 369 patients diagnosed with PD/A. The MAC study utilized a multi-center, randomized controlled design, with two active treatment conditions in which the administration of exposure was varied, and a wait-list control group. The special feature of MAC is the way in which imbedded experimental, psychophysiological, and neurobiological paradigms were included to elucidate therapeutic and psychopathological processes. This paper describes the aims and goals of the MAC study and the methods utilized to achieve them. All aspects of the research design (e.g., assessments, treatment, experimental procedures) were implemented so as to facilitate the detection of active therapeutic components, and the mediators and moderators of therapeutic change. To this end, clinical, behavioral, physiological, experimental, and genetic data were collected and will be integrated.

Tissue healing is in general a complex process, which involves both local and systemic responses, and bone regeneration in particular is much slower than repair in any other human tissue. Thus, it exhibits a great challenge in clinical practice and in the field of research. Bone regeneration is comprised of a series of biological events, involving a number of cell types and intracellular and extracellular molecular- signaling pathways, with a definable temporal and spatial sequence, in an effort to optimize the skeletal repair and restore its functionality. Photobiomodulation (PBM) therapy has been shown to be effective in modulating both local and systemic responses, by enhancing cellular activities resulting in an increase in function, especially in injured tissues, leading to optimization of tissue repair and regeneration. In bone tissue, application of the photonic energy leads to bone healing by the activation of osteoblasts, leading to proliferation and differentiation, as well as osteoclast inhibition and, consequently, neoformation of bone matrix. The process of the in vitro pre-osteoblasts maturation, mimicking their in vivo behavior, passes through three distinct stages of development: proliferation, early differentiation (maturation) and late differentiation (mineralization). Despite the extensive research on the effects of photobiomodulation (PBM) light on bone regeneration, the current outcomes ranging from positive to negative effect remain controversial. These contradictory data are thought to be due to; incomplete knowledge and understanding of the mechanistic effects of laser light on cells, lack of standardized laser dosimetry, inefficient laser beam profile, improper study design and varied methods of investigation. The literature is hindered by a considerable heterogeneity of the irradiation parameters of PBM, as well as, the methods utilized to evaluate the results and the type of osteoblast-like cells irradiated. This has led to a need of standardization. Moreover, heterogeneity of the current studies and their limitations could be due to study designs and inefficient beam profile, resulting in undesirable effects and accounting for negative and inconclusive outcomes. Ultimately, lack of intimate knowledge and understanding of the PBM light behavior impinging on the target tissue, as well as the optical tissue properties, can compromise optimization of the therapeutic outcomes. Thus, an evidence-based decision for definite therapeutic application of PBM in bone regeneration is required. In this thesis, we addressed the above issues and challenges via two elements, the electromagnetic (EM) modeling experiments and the molecular and cellular impact of PBM on bone regeneration (In vitro studies). The Electromagnetic models In my PhD proposal, I intended to both create an EM model, for the first time, and examine the mechanism of interaction of the electromagnetic fields (EMFs) with cells/tissues and establish the link that can be utilized in my cellular experiments. As the project evolved, it became clear this work was breaking new grounds and was significantly more complex than initially envisaged. As it is a small part of a much larger exciting project undertaken by University of Genoa, it has meant that I need to coordinate my work with the overall timetable of this larger project. As a result, I decided to defer, the interaction of the EMF with cells of interest part, to my Post doctorate study. We developed, for the first time, a set of simple models to examine the behavior of the local electromagnetic field (EMF), determining the PBM effects on mitochondria. This set of models was tested and crosschecked for its validity by evaluating various variables in terms of, polarization, absorption and scattering coefficient, dissipated energy density and irradiance, as well as the refractive index. Ultimately, our model and preliminary data are the first stepping-stone for further experiments, in order to understand the mechanism of interaction between electromagnetic fields and cells or tissues. Our conclusions showed that when these set of models are utilized, for the phenomenon of interest, the incident field polarization had small effects on the electromagnetic field and negligible consequences on the average energy, as well as, on the dissipated power densities. The same was shown to hold true for different orientations that the mitochondria can assume. The analogous conclusions were obtained by taking into account the possible changes in the dimensions or of the real part of the refractive index of the considered organelles. The variations of the absorption coefficient were shown to have significant effects on the average dissipated power density in the mitochondria but these effects can be predicted in a surprisingly simple way. It was proved that the numerical analysis, of the problems of interest, could be computed by using three-dimensional models, involving only a few mitochondria in the plane, which was transverse to the direction of propagation of the illuminating light that generated a uniform distribution of the energy over 1cm2 area. The one- dimensional models provided significant information on the EMF, utilized to stimulate the mitochondria. Mitochondria behaved like weak scatterers. Therefore, it was not necessary to analyze large extension of such organelles to understand what happen inside one of them. The molecular and cellular impact of 980nm PBM on osteoblast maturation: in vitro studies Our pilot study data, on the bone marrow stromal cells (BMSCs), strongly suggested that the high fluence concept (over 60J/cm2 in continuous emission mode (CW)) delivered by flattop beam profile device (FT) can promote BMSCs differentiation towards osteogenesis. Moreover, the results showed an increase in cytokines synthesis with potent anti-inflammatory properties and a decrease in the release of proinflammatory mediators. This provided me with a platform, demonstrating the validity of high fluence in facilitating osteoblasts differentiation through BMSCs. Based on this; I formulated three PBM protocols for 980nm to be tested on pre- osteoblast cell line in my definitive in vitro studies. The first phase of in vitro studies aimed to evaluate the 980nm bio-stimulatory effects on osteoblasts maturation, optimise the PBM effects on bone healing with various beam profiles delivery devices, and establish protocol/protocols of 980nm PBM in bone regeneration. The primary objective was to determine the optimal 980nm dosimetry, which exerts bio- stimulatory effects to accelerate and enhance the bone regenerative process. The secondary objective was to evaluate the intra-cellular pathways of the photon-cell interaction across the metabolic proliferative and differentiation changes, which ultimately lead to bone healing and repair. The results of this study validated the contribution of PBM in bone regeneration and elucidated the biochemical effects at a cellular level. Moreover, the role of different dosages of 980nm PBM irradiation delivered by FT; in comparison to the Gaussian beam profiles (Standard (ST)) on bone regeneration were highlighted. The setup of the power outputs on the laser device was 1.1Watt (W) for the ST and 1W for the FT. However, the real (the threshold) power output reaching the target, measured by power meter, was as ∼0.9 W, (Irradiance ∼ 0.9W/cm2, Exposure time 60 seconds, energy ∼55 J (Joule), fluence ∼55 J/cm2) delivered with the FT beam profile in CW in comparison to the ST, on MC3T3-E1 pre-osteoblast maturation. The protocol was based on 60 seconds exposure time for two consecutive weeks, which employed for all the groups. The laser grouping and their associated irriadtied energies were as follows: Group 1- Irradiation once per week (Total enrgy 110J). Group 2- Irradiation three times per week (Alternate day) (Total energy 330J). Group 3 - irradiation five- times per week (Total energy 550 J). The control cultures were processed in identical conditions except that the laser device was kept off all the time. The total energy was 0J.
The metabolic activity and the osteoblasts maturation were analyzed by alkaline phosphatase assay, alizarin red S histological staining, immunoblot and/or double immunolabeling analysis for Bcl2, Bax, Runx-2, Osx, Dlx5, osteocalcin, and collagen Type 1. Our data, for the first time, prove that laser irradiation of 980 nm wavelength with flattop beam profile delivery system, compared to standard-Gaussian profile, has improved photobiomodulatory efficacy on pre-osteoblastic cells differentiation. Mechanistically, the irradiation enhances the pre-osteoblast differentiation through activation of Wnt signaling as well as the Smads 2/3-βcatenin pathway. Our results indicated and valued the intra-cellular pathways of the photon-cell interaction across the metabolic, proliferative and differentiation changes in the cells. Additionally, our data showed that the cells irradiated THREE times a week (Total energy of 330 J) and ONCE a week (Total energy of 110 J) for two consecutive weeks protocols have increased the proliferation and differentiation of the osteoblasts in both ST & FT hand-pieces but the data showed increasingly statistical significant in the FT group. The only Runx2 was detected when the cells were irradiated with the ST hand-piece. Therefore, total energy of 110 J when either of the hand-pieces utilized, has influenced early differentiation markers. Interestingly, when the process was carried out, until the mineralization and maturation (Late osteogenesis), the ST hand-piece irradiation failed to induce an effective process, and did not lead to matrix deposition, while the FT profile showed a significant effect. In conclusion, our data, for the first time, prove that laser irradiation of 980 nm wavelength with the FT beam profile delivery system in comparison to the ST profile has a great photobiomodulatory efficacy on pre-osteoblastic cells differentiation, which would assist in accelerating bone regeneration, due to its homogeneous energy distribution at each point of its cross-section. Moreover, the irradiation protocols of three times a week and once a week for two consecutive weeks were able to increase the pre-osteoblasts and osteoblasts transcription factors, which were strongly and statistically significantly increased when the FT hand-piece was utilized. Therefore, the 980 nm laser irradiation protocol was able to promote the MC3T3-E1 cell differentiation. Researchers have demonstrated that the major barrier for an effective biological healing is insufficient laser photonic energy delivered to the injured site. PBM can modify the cell metabolism by increasing the mitochondria's ATP production. Currently, the challenge is to understand the target tissues optical properties and its cellular pathway when irradiated with laser phonic energy. In this way, modification of various energy exposure values can influence clinical outcomes predictability. Therefore, in the second phase of my in vitro study, we evaluated the effect of 980nm irradiation delivered with ST and FT beam profile hand-pieces on monolayer cell, at various power outputs; 0.8W, 0.5W and 0.25W. However, the exact power output values reaching the target, measured by power meter, were as follows: 0.75W, 0.45W, and 0.20W respectively. The MC3T3-E1 cells irradiated for two consecutive weeks, according to the following protocols: once a week (Total energy 90, 54, 24 J), respectively); three times a week (total energy 270, 162, 72J, respectively); five times a week (total energy 450, 270, 120 J, respectively). Metabolic activity of viable cells evaluated as follows: Hoechst staining; Western blotting for Runx-2, Bcl2, Bax, Osx, Dlx5, β-catenin, Smads 2/3, TGFβ, p.PI3K, PI3K, p.AKt, AKt, and p.ERK. Our data, for the first time, prove that the 980 nm irradiation at power output setting at 0.75W (0.75W/cm2) for 60 seconds in CW stimulated the MC3T3-E1 pre- osteoblasts viability, by affecting the critical pre-survival markers such as p.PI3K, p.Akt, Bcl2 and Bclxl. Moreover, we concluded that 980nm PBM delivered with FT at 0.75W power output was comparable to results with the ST. However, 0.45W and 0.20W did not modulate the cell metabolic features. Additionally, none of the laser protocols delivered with FT or ST had any influence on the cell differentiation process. In summary, our in vitro studies data, for the first time, have demonstrated the potential of utilizing the FT beam profile with our established protocols in bone regeneration, as a therapeutic tool for future pre-clinical and clinical applications. Moreover, these studies have shown the mechanistic effects of the PBM light on intracellular pathway across the metabolic and differentiation of the osteoblasts towards bone regeneration.

INTRODUCTION: Major depressive disorder (MDD) is characterized by ongoing feelings of guilt, sadness, and memory and cognition impairment. It is a multidimensional illness that affects many functionally integrated pathways of the brain. Understanding the underlying brain dysfunction that gives rise to this complex illness has been challenging, and by extension the search for appropriate treatments. MDD patients who are considered treatment resistant make up the primary population that receives electroconvulsive therapy (ECT). Remarkably, ECT shows a 75% remission rate in this patient population and is considered the “gold standard” treatment for major depression. Although the exact mechanism of its function is unknown, it is well accepted that the induced grand-mal seizure confers its therapeutic effect. The seizure likely has broad effect that somehow corrects the underlying dysfunction in brain circuitry. Here, we specifically examined studies of functional connectivity and metabolite changes. METHODS: Through literature search, we examined six studies in functional connectivity and four studies in magnetic resonance spectroscopy (MRS). RESULTS: Functional Connectivity: Studies have found that after bilateral ECT treatments, patients with major depression showed reduction of functional connectivity (FC) from the left dorsolateral prefrontal cortex (DLPFC) to other cortical and limbic structures. Correlated activity between the superior frontal gyri, middle frontal gyri and angular gyri were significantly increased after ECT. Hyperdeactivation of the orbitofrontal cortex to negative emotional stimuli in patients was decreased, and it was associated with improvement in depressive symptoms. Regional activity in the subgenual anterior cingulate cortex (sgACC) and functional connectivity between the sgACC and left hippocampus in treatment naïve patients after ECT were increased and correlated to reduction of depressive symptoms. Reduced connectivity between the amygdale and sgACC and increased connectivity between the amygdale and DLPFC was found by sequential assessments over a course of ECT treatments. Lastly, ECT increased the functional connectivity between DLPFC and the default mode network. MRS: Studies found decreased levels of glutamate or glx (glutamate/glutamine/ GABA) in patients in the anterior cingulate cortex and dorsolateral prefrontal cortex (DLPFC) compared to healthy controls. Additionally, it was found that glx levels increased after ECT treatments and that this increase was only in those who responded to treatment. Lastly, GABA level increased after ECT treatment in the occipital cortex. Discussion: Results from functional connectivity and brain metabolite studies in patients with major depression point to induced neuroplasticity as part of ECT’s therapeutic mechanism. Remodeling connectivity and mediating metabolite changes both will require modifications at the synaptic level. The wide spread changes seen in several different brain regions that have been implicated in depression further suggests that ECT’s effects are both highly specific and broad. CONCLUSION: Electroconvulsive therapy has consistently demonstrated impressive efficacy among the most severely depressed patients and is known to produce widely distributed effects in the brain. However, this also makes assessing its therapeutic mechanism challenging. Magnetic resonance imaging studies assessing functional connectivity and brain metabolite levels have demonstrated that ECT likely produces neuroplastic changes to remodel aberrant connectivity and dysfunctional excitatory and inhibitory neurotransmission in cortical and limbic areas. Although these findings should be interpreted with caution, this field of research has provided an unprecedented opportunity to examine the living brain in great detail. Further studies with larger sample sizes and improved technical specifications will likely yield greater results.

碩士
國立清華大學
生物資訊與結構生物研究所
104
Boron neutron capture therapy (BNCT) is a targeted radiation therapy under clinical trial in Taiwan. Boron-10 delivered into the tumor cells disintegrates after capturing a neutron, and the high energy heavy charged particles produced from the nuclear event destroy cancer cells, with only marginal damage to the surrounding normal tissues. BNCT has been used in clinical trials to treat various types of cancers. In 2009, Prof. Fong-In Chou of National Tsing Hua University found, in addition to borophenylalanine (BPA) and borocaptate (BSH), boric acid (BA) could also be the boron carrier for BNCT. Specifically, boric acid could be a promising agent for BNCT treatment of hepatocellular carcinoma (HCC). However, the underlying mechanism that influence tumor to normal tissue (T/N) ratio of the BA uptake and the transport process is still unclear. There are numerous reports showing that tumor microenvironment is different from the surrounding normal cells, including the glycoprotein profile, glucose metabolism and microenvironment pH value. Other reports also show that BA could form complexes with carboxylates, which involve covalent interactions that are reversible in aqueous solution. For these reasons, we hypothesized the differences between tumor and normal cells microenvironment may cause differential uptake of boron/BA into the liver cancer cells prior neutron irradiation. Consequently, our experimental data implied glucose metabolic condition and acidic environment might regulate the uptake of boron/BA by the liver cancer cells. Meanwhile, in order to accelerate the preclinical study of BA-mediated BNCT in established a zebrafish xenograft model with mouse melanoma cell line B16-F10 and adapted a transgenic zebrafish line that could develop spontaneous melanoma. We demonstrated that zebrafish could absorb boron in the delivery form of BA by orbital injection. A holding device was developed to immobilize the zebrafish during neutron irradiation. This novel zebrafish BNCT platform shall help us to understand the BA-mediated BNCT mechanism and speed up its translational application in the future.

Pang Siu Kwong.
"August 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 181-191)
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

Sam Sze Wing.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.
Includes bibliographical references (leaves 156-184).
Abstracts in English and Chinese.
Publications based on work in this thesis --- p.ii
Abstract --- p.iii
Acknowledgements --- p.vii
Chapter 1 --- INTRODUCTION --- p.1
Chapter 1.1 --- Corticosteroids --- p.2
Chapter 1.1.1 --- Chemical Structure of Steroids --- p.3
Chapter 1.1.2 --- Biosynthesis of Endogenous Corticosteroids --- p.3
Chapter 1.1.2.1 --- Regulation of Cortisol synthesis and negative feedback system --- p.4
Chapter 1.1.3 --- Biological Significance of Corticosteroids --- p.5
Chapter 1.1.3.1 --- Involvement of corticosteroids as anti-inflammatory drugs --- p.6
Chapter 1.1.3.2 --- Eicosanoid biosynthesis --- p.7
Chapter 1.1.3.3 --- Lipoxygenase pathway --- p.9
Chapter 1.1.3.4 --- Side-effects of prolonged use of corticosteroids --- p.9
Chapter 1.2 --- Organisation of the Emetic Reflex --- p.11
Chapter 1.2.1 --- Motor Pathway of Emetic Reflex --- p.12
Chapter 1.2.1.1 --- Retching and vomiting --- p.12
Chapter 1.2.1.2 --- Nausea --- p.13
Chapter 1.2.2 --- Components of the Emetic Reflex --- p.14
Chapter 1.2.2.1 --- The vomiting centre (VC) --- p.15
Chapter 1.2.2.2 --- Area postrema (AP) / Chemoreceptor trigger zone (CTZ) --- p.15
Chapter 1.2.2.3 --- The nucleus tractus solitarius (NTS) --- p.17
Chapter 1.2.2.4 --- Gastrointestinal tract and vagus nerves --- p.17
Chapter 1.2.2.5 --- Neurotransmitter receptors --- p.18
Chapter 1.3 --- Chemotherapy-Induced Emesis --- p.19
Chapter 1.3.1 --- Cancer as a cause of mortality in Man --- p.20
Chapter 1.3.2 --- Chemotherapeutic Agents --- p.20
Chapter 1.3.2.1 --- Different classes --- p.20
Chapter 1.3.2.2 --- Emetogenic potential --- p.21
Chapter 1.3.3 --- Cisplatin-Induced Emesis --- p.23
Chapter 1.3.3.1 --- Unfavourable effects associated with chemotherapy-induced nausea and emesis --- p.24
Chapter 1.3.3.2 --- Anticipatory nausea and vomiting --- p.24
Chapter 1.3.3.3 --- Profile of cisplatin-induced emesis --- p.25
Chapter 1.3.4 --- Animal Models of Cisplatin-Induced Acute and Delayed Emesis --- p.26
Chapter 1.3.5 --- Mechanisms and Pathways Involves in Chemotherapy-Induced Emesis --- p.28
Chapter 1.3.6 --- Anti-Emetic Drugs for the Treatment of Chemotherapy-Induced Emesis --- p.31
Chapter 1.3.6.1 --- 5-HT3 receptor antagonists --- p.31
Chapter 1.3.6.2 --- Dopamine receptor antagonists --- p.33
Chapter 1.3.6.3 --- Benzodiazepines --- p.35
Chapter 1.3.6.4 --- Cannabinoids --- p.35
Chapter 1.3.6.5 --- Antihistamines and anticholinergics --- p.35
Chapter 1.3.6.6 --- NK1 receptor antagonists --- p.37
Chapter 1.3.6.7 --- Corticosteroids --- p.38
Chapter 1.3.6.8 --- Multi-agent anti-emetic regimens --- p.39
Chapter 1.4 --- Motion-Induced Emesis --- p.41
Chapter 1.4.1 --- Incidence --- p.42
Chapter 1.4.2 --- Mechanisms and Pathways Involved in Motion Sickness --- p.43
Chapter 1.4.2.1 --- Importance of the vestibular apparatus --- p.44
Chapter 1.4.2.2 --- Importance of the area postrema --- p.45
Chapter 1.4.2.3 --- The nucleus tractus solitarius --- p.46
Chapter 1.4.2.4 --- Hormone and neurotransmitters --- p.46
Chapter 1.4.3 --- Animal models in Motion-Induced Emesis --- p.47
Chapter 1.4.4 --- Anti-Emetic Drugs for the Treatment of Motion Sickness --- p.48
Chapter 1.4.4.1 --- Anticholinergics --- p.49
Chapter 1.4.4.2 --- Antihistamines --- p.49
Chapter 1.4.4.3 --- Non-selective muscarinic and histamine receptor antagonists --- p.51
Chapter 1.4.4.4 --- Sympathomimetics --- p.51
Chapter 1.4.4.5 --- NK1i receptor antagonists --- p.51
Chapter 1.4.4.6 --- 5-HT1A agonists --- p.52
Chapter 1.4.4.7 --- 5-HT2 receptor agonist --- p.52
Chapter 1.4.4.8 --- Arginine vasopressin (AVP) antagonists --- p.53
Chapter 1.4.4.9 --- Opioid receptor agonists --- p.53
Chapter 1.4.4.10 --- Dexamethasone and hormone levels --- p.54
Chapter 1.4.4.11 --- Other anti-emetic drugs --- p.55
Chapter 1.5 --- Aims of the Studies --- p.56
Chapter 2 --- Methods --- p.59
Chapter 2.1 --- Cisplatin-Induced Emesis Studies --- p.60
Chapter 2.1.1 --- Animals --- p.60
Chapter 2.1.2 --- Induction and Measurement of Emesis --- p.60
Chapter 2.1.3 --- The Effects of Corticosteroids on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.63
Chapter 2.1.4 --- "The Effects of Dexamethasone (1 mg/kg, i.p.) Administered as an Intervention Treatment on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.63
Chapter 2.1.5 --- The Effects of Cortrosyn Depot (Tetracosactrin) on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.63
Chapter 2.1.6 --- The Effects of Metyrapone on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.64
Chapter 2.1.7 --- The Effects of Indomethacin on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.64
Chapter 2.1.8 --- "The Effects of DFU and L-745,337 Administered as an Intervention Treatments on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.64
Chapter 2.1.9 --- "The Effects of MK-886 (L-663,536) on Cisplatin-Induced Acute and Delayed Retching and Vomiting" --- p.65
Chapter 2.1.10 --- The Effects of a Combination of Indomethacin and MK-886 on Cisplatin- Induced Acute and Delayed Retching and Vomiting --- p.65
Chapter 2.1.11 --- Statistical Analysis --- p.66
Chapter 2.2 --- Motion-Induced Emesis Studies --- p.67
Chapter 2.2.1 --- Animals --- p.67
Chapter 2.2.2 --- Measurement of Emesis --- p.67
Chapter 2.2.3 --- Induction of Emesis in Motion-Naive Suncus murinus: Effects of Glucocorticoids --- p.68
Chapter 2.2.4 --- Induction of Emesis in Motion-Sensitive Suncus murinus: Effects of Dexamethasone --- p.70
Chapter 2.2.5 --- Preparation of Serum --- p.72
Chapter 2.2.6 --- Measurement of Serum Cortisol by Enzyme-Linked Immunoassay (ELISA) --- p.72
Chapter 2.2.6.1 --- Immunoassay kit --- p.72
Chapter 2.2.6.2 --- Assay procedures --- p.73
Chapter 2.2.7 --- Measurement of Serum Adrenocorticotrophin (ACTH) by Radioimmunoassay (RIA) --- p.75
Chapter 2.2.7.1 --- Immunoassay kit --- p.75
Chapter 2.2.7.2 --- Assay procedures --- p.76
Chapter 2.2.8 --- Statistical Analysis --- p.79
Chapter 3 --- Results --- p.81
Chapter 3.1 --- Cisplatin-Induced Emesis --- p.82
Chapter 3.1.1 --- General Profile of Emesis Induced by Cisplatin --- p.82
Chapter 3.1.2 --- Antagonism of Cisplatin-Induced Emesis by Corticosteroids --- p.82
Chapter 3.1.3 --- "The Effect of Dexamethasone (1 mg/kg, i.p.) Administered as an Intervention Treatment on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.84
Chapter 3.1.4 --- The Effect of Cortrosyn Depot (Tetracosactrin) on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.85
Chapter 3.1.5 --- The Effect of Metyrapone on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.85
Chapter 3.1.6 --- "The Effect of Indomethacin, DFU and L-745,337 on Cisplatin-Induced Acute and Delayed Retching and Vomiting" --- p.86
Chapter 3.1.7 --- The Effect of MK-886 on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.88
Chapter 3.1.8 --- The Effect of Combination of Indomethacin and MK-886 on Cisplatin- Induced Acute and Delayed Retching and Vomiting --- p.89
Chapter 3.2 --- Motion-Induced Emesis --- p.91
Chapter 3.2.1 --- General Effect of Motion on Serum Cortisol and ACTH Levelsin Motion Naive Suncus murinus --- p.91
Chapter 3.2.2 --- The Effect of Glucocorticoids on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion-Naive Male Suncus murinus --- p.92
Chapter 3.2.2.1 --- Effect of dexamethasone --- p.92
Chapter 3.2.2.2 --- Effect of betamethasone --- p.93
Chapter 3.2.2.3 --- Effect of methylprednisolone --- p.93
Chapter 3.2.3 --- The Effect of Glucocorticoids on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion Naive Female Suncus murinus --- p.94
Chapter 3.2.3.1 --- Effect of dexamethasone --- p.94
Chapter 3.2.3.2 --- Effect of betamethasone --- p.95
Chapter 3.2.3.3 --- Effect of methylprednisolone --- p.95
Chapter 3.2.4 --- The Effect of Dexamethasone on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion-Sensitive Suncus murinus --- p.96
Chapter 3.2.4.1 --- Effect of dexamethasone on male motion-sensitive animals --- p.97
Chapter 3.2.4.2 --- Effect of dexamethasone on female motion-sensitive animals --- p.97
Chapter 4 --- Discussion --- p.131
Chapter 4.1 --- "Cisplatin (5 mg/kg, i.p.)-Induced Emesis in Control Animals" --- p.132
Chapter 4.2 --- Anti-Emetic Action of Corticosteroids in the Ferret --- p.133
Chapter 4.3 --- Metyrapone Study --- p.138
Chapter 4.4 --- Cortrosyn Depot Study --- p.139
Chapter 4.5 --- Role of Cycloxygenase --- p.141
Chapter 4.6 --- Role of 5-Lipoxygenase --- p.143
Chapter 4.7 --- Duel Inhibition of Cycloxygenase and 5-Lipoxygenase --- p.144
Chapter 4.8 --- Anti-Emetic Potential of Glucocorticoids in Suncus murinus --- p.145
Chapter 4.9 --- General Summary --- p.149
Appendix I --- p.152
Appendix II --- p.154
References --- p.156

Despite advances in technology and understanding of biological systems in the past two decades, modern drug discovery is still a lengthy, expensive, difficult and inefficient process with low rate of new therapeutic discovery. The search for new effective drugs remains a somewhat empirical process. There is compelling need for a more fundamental, mechanistic understanding of human cancers and anticancer drugs to design more appropriate drugs. Radiotherapy is still the major therapy of cancer. It uses high-energy ionizing radiation such as x-rays and charged particle beams to destroy cancer cells. DNA is well known to be the principal biological target of radiotherapy, but the molecular mechanism of ionizing radiation induced DNA damage was elusive. The conventional thought of the ???OH radical as the major origin for ionizing radiation induced DNA damage is questionable. Although various strategies and types of compounds have been designed and developed as potential radiosensitizers to enhance the radiosensitizing efficiency of radiotherapy, none of them have been approved for clinical use. The general outcomes of clinical trials have been disappointing. This thesis presents an innovative molecular-mechanism-based drug discovery project to develop novel drugs for effective radiotherapy of cancer through the emerging femtomedicine approach. Its ultimate goal is to develop more effective radiosensitizers, based on our unique molecular understandings of ionizing radiation induced DNA damage and halopyrimidines as a family of potential radiosensitizers. Direct, real-time observation of molecular reactions is of significant importance in diverse fields from chemistry and biology, environmental sciences to medicine. Femtosecond time-resolved laser spectroscopy (fs-TRLS) is a very powerful, direct technique for real-time observation of molecular reactions. Its key strength lies in short duration laser flashes of a time scale at which reactions actually happen - femtoseconds (fs) (1fs = 10???15 second). Since the late 1980s, its application to study chemical and biological systems led to the births of new subfields of science, called femtochemistry and femtobiology. Recently, femtomedicine has been proposed as a new transdisciplinary frontier to integrate ultrafast laser techniques with biomedical methods for advances in fundamental understandings and treatments of major human diseases. This the remarkable opportunity afforded through real-time observation of biochemical reactions at the molecular level. Femtomedicine holds the promise of advances in the radiotherapy of cancer. Several important findings were made in this thesis. First, our results of careful and high-quality fs-TRLS measurements have resolved the long existing controversies about the physical nature and lifetimes of a novel ultrashort-lived electron species (epre???) generated in radiolysis of water. These results have not only resolved the large discrepancies existing in the literature but provided new insights into electron hydration dynamics in bulk water. Such information is important for quantitative understanding and modeling of the role of non-equilibrium epre??? in electron-driven reactions in diverse environmental and biological systems, from radiation chemistry and radiation biology to atmospheric ozone depletion. Second, our fs-TRLS results have unraveled how epre??? plays a crucial role in ionizing radiation induced DNA damage. We found that among DNA bases, only T and especially G are vulnerable to a dissociative electron transfer (DET) reaction with epre??? leading to bond breaks, while the electron can be stably trapped at C and especially A to form stable anions. The results not only challenge the conventional notion that damage to the genome by ionizing radiation is mainly induced by the oxidizing ???OH radical, but provide a deeper fundamental understanding of the molecular mechanism of the DNA damage caused by a reductive agent (epre???). Our findings have led to a new molecular mechanism of reductive DNA damage. Third, halopyrimidines, especially BrdU and IdU, have passed Phase I to II clinical trials as potential hypoxic radiosensitizers, but the outcome of Phase III clinical trials was disappointing. Our results of fs-TRLS studies have provided a new molecular mechanism of action of halopyrimidines (XdUs, X=F, Cl, Br and I) in liquid water under ionizing radiation. We found that it is the ultrashort-lived epre???, rather than the long-lived ehyd???, that is responsible for DET reactions of XdUs. This reaction leads to the formation of the reactive dU??? radical, which then causes DNA strand breaks and cancer cell death. Our results have challenged a long accepted mechanism that long-lived ehyd??? would be responsible for the radical formation from halogenated molecules. Furthermore, we found that the DET reaction efficacy leading to the formation of the reactive dU??? radical is in the order of FdU << CldU < BrdU < IdU. Thus, only BrdU and IdU could be explored as potential radiosensitizers, in agreement with the results of bioactivity tests and clinical trials. Fourth, our fs-TRLS studies have provided a molecular mechanism for the DNA sequence selectivity of BrdU and IdU in radiosensitization. We found the DET reactions of BrdU/ IdU with dAMP*??? and dGMP*??? formed by attachment of epre??? generated by radiolysis of water in aqueous BrdU-dAMP/dGMP and IdU-dAMP/dGMP complexes under ionizing radiation. This new mechanistic insight into the interaction of BrdU and IdU with DNA provides clues to improve the halogen familty as potential radiosensitizers and to develop more effective radiosensitizers for clinical applications. Fifth, based on our molecular mechanistic understandings of DNA damage induced by ionizing radiation and halopyrimidines as potential radiosensitizers, we develop more effective new radisensitizing drug candidates through the femtomedicine approach. We have performed a fs-TRLS study of the DET reaction of a candidate compound (RS-1) with epre???, and found that the DET reaction of epre??? with RS-1 is much stronger than that of IdU (and certainly BrdU and CldU). Moreover, we have tested the radiosensitizing effect of RS-1 against human cervical cancer (HeLa) cells exposed to various doses of x-ray irradiation through DNA damage measurements by gel electrophoresis and cell viability/death assays by MTT. Our results have confirmed that RS-1 can largely enhance the radiosensitivity of treated human cervical cancer (HeLa) cells to x-ray (ionizing) radiation. It is clearly demonstrated that RS-1 has a much better radiosensitizing effect than IdU. Although these are just preliminary results, our results have shown promise of developing more effective radiosensitizers. In summary, our studies have demonstrated the potential of femtomedicine as an exciting new frontier to bring breakthroughs in understanding fundamental biological processes and to provide an efficient and economical strategy for development of new anticancer drugs.

While the molecular defect that cause sickle cell disease has well been established, the cause of vaso-occlusive crisis remains elusive and largely debated upon. Majority of studies have linked the painful episodes to polymerization of sickle hemoglobin following its deoxygenation. The variability of the disease symptoms among patients, compounds efforts for a holistic therapy. Hydroxyurea, a stimulator of Hb F induction and a widely used treatment, has ameliorated the complication of SCD but it is only effective in 50% of the patients. Expression of Hb F lowers the content of Hb S in blood and hence reduces oxidative stress caused by Hb S denaturation. Sickle cell disease severity depends on several factors. Most importantly, the ability of red cell to sickle dominates all other determinants. While deoxygenation of sickle hemoglobin may be inevitable, the duration with which the red cell remains in the deoxygenated state can be manipulated. Deoxygenation is a transient process that when compared to the time taken to develop the long filaments of deoxyhemoglobin to causes severe sickling, the red cell would have been cycled back to the lungs and re-oxygenated to restore the healthy conditions of the cell. In fact, if sickle cells would flow as fast as healthy erythrocytes, the detrimental impacts of sickling such as vaso-occlusive crisis, would not be a concern for this disease. Unfortunately, the unstable sickle hemoglobin undergoes denaturation through auto-oxidation, which imposes oxidative stress to the cells. The oxidative stress inhibits erythrocytes tyrosine phosphatases, a course which subsequently impair their constitutive action against the tyrosine kinases. In the end, a net tyrosine phosphorylation state in the red cell membrane proteins, most notably the transmembrane protein band 3, succeeds. Band 3 tyrosine phosphorylation abrogates the protein’s interaction with ankyrin and spectrin-actin cytoskeleton, hence the cytoskeleton loses its major anchorage to the membrane thus engendering membrane destabilization. A destabilized erythrocyte sheds membrane fragments in form of microvesicles/microparticles and discharges free hemoglobin into the extra cellular matrix. In consequence, the microparticles power initiation of coagulation cascade through activation of thrombin, while free Hb inflicts inflammation, scavenges nitric oxide which is necessary for vasodilation and induces further oxidative stress within the microvasculature, and activates expression of adhesion receptors on the endothelium. Taken together, these events culminate in entrapment of red cells (not naming leucocytes and platelets) in the microvasculature, blockade of blood vessels and further damage of erythrocytes through prolonged deoxygenated state thus terminating in tissue injury, strokes, and organ damage, amid vaso-occlusive episodes which always require hospitalization and extensive medical care for survival. Band 3 tyrosine phosphorylation and membrane weakening is not unique just to SCD, but also a druggable target for malaria. Malaria, a disease that is touted as the evolutionary cause of sickle cell disease, surprisingly thrives through the same mechanism. Briefly, malaria parasite consumes hemoglobin for its DNA synthesis, and in the process generate reactive oxygen species from denatured hemoglobin that feeds into the oxidative stress which triggers band 3 tyrosine phosphorylation. In this case however, a destabilized membrane offers perfect conditions for merozoites’ (malaria daughter parasites) egress/exit out of the cell to begin infecting other red cells. Ultimately, the ensuing anemia and organ dysfunction leads to patient’s death. Treatment of diseased cells with imatinib and other Syk inhibitors effectively reversed membrane weakening. A stabilized membrane not only survives longer in circulation to alleviate SCD symptoms but also traps and starves malaria parasite leading to termination of the parasitic infection. With band 3 tyrosine phosphorylation at center stage, this dissertation explores the above events in an effort to unveil a novel therapy for sickle cell and malaria diseases. First, the therapeutic strategy regarding SCD is discussed in detail beginning with non-transfused patients and ending in additional mechanistic study on inactivation of the principal erythrocyte’s protein tyrosine phosphatase 1 B, PTP1B. The dissertation then provides an initial proof of concept on efficacy of imatinib in treatment of malaria as a monotherapy and its efficacy when used in a triple combination therapy with the standard of care treatment. Finally, I outline an alternative possible mechanism of action of quinine against malaria.