Dissertations / Theses on the topic 'HDIC'

2012/2013
La tecnologia dei sistemi micro-elettro-meccanici (MEMS) ha dimostrato d’avere grandi potenzialità in molti campi, in particolare nei sistemi bio-medicali. Essa si basa infatti su processi di fabbricazione ad altro volume produttivo, permettendo una considerevole riduzione dei costi per dispositivo. Un ulteriore beneficio di questa tecnologia risiede nella possibilità di dimensionare i dispositivi fino a raggiungere l’ordine del submicron, così da consentire l’integrazione e il monitoraggio in tempo reale di sistemi sensibili a biomarker di tipo medicale e biologici. Tra gli obiettivi futuri dei MEMS biomedicali (BioMEMS) vi è la realizzazione di dispositivi in grado di interfacciarsi direttamente con il paziente e definirne lo stato di salute grazie alla rilevazione del livello di centinaia di diversi biomarker (siano essi chimici o fisici). La medicina assumerebbe in questa visione una configurazione ad personam nella quale al paziente verrebbe prontamente somministrato un quantitativo di medicinale adatto alle risposte del suo organismo. A tale scopo i dispositivi MEMS devono essere in grado di effettuare analisi multiple operando in un ambiente liquido. Tuttavia è proprio l’ambiente liquido a comportare la riduzione di sensibilità e, quindi, di performance dei sensori MEMS. La presente ricerca si pone lo scopo di sviluppare nuovi sistemi elettronici di misurazione e attuazione di due distinte tipologie di BioMEMS risonanti operanti in liquido, i cantilever e i pillar. In particolare verrano trattati tre argomenti: la realizzazione di setup ottici per applicazione dei MEMS in liquido ed in aria, la progettazione di sistemi elettronici di attuazione e lettura di singoli pillar nel loro comportamento in frequenza e lo sviluppo di un software LabVIEW in grado di programmare un FPGA ed ottenere un PLL digitale da impiegarsi nell’analisi in tempo reale del comportamento in frequenza di RF-MEMS. Il primo progetto è stato sviluppato in collaborazione l'Università di Kaiserslautern (Germania) e prevedeva la realizzazione di sistemi microfluidici e setups ottici, interfacciati in modo tale da permettere la rilevazione della risposta in frequenza di molteplici MEMS operanti in parallelo. Nel secondo progetto l’obiettivo era la realizzazione di un sistema elettronico in grado di integrare in un unico dispositivo i sistemi di attuazione e lettura dei pillar. In particolare siamo stati in grado di modulare l’ampiezza di risonanza dei nostri dispositivi risonanti mediante l’applicazione della forza di polarizzazione Kelvin mentre lo sviluppo del sistema di lettura richiede ulteriore lavoro di indagine. Infine, nell'ultimo progetto è stato realizzato un sistema PLL digitale con 10 MHz di banda passante utilizzando la tecnologia della National Instruments (FlexRIO NI5781R). Mediante questo PLL si è potuto identificare la frequenza di risonanza di diverse tipologie di MEMS e se ne è seguite le variazioni in tempo reale . Le attività di ricerca sperimentale sono state eseguite presso il laboratorio CNR- IOM a Trieste.
XXVI Ciclo
1985

Il controllo epigenetico dell'espressione genica e' operato da modifiche post-translazionali della cromatina, quali acetilazione e deacetilazione. Due enzimi sono respinsabili di questo processo: le HAT (istone acetiltransferasi) e le HDAC (istone deacetilasi). Le HDAC sono un gruppo di enzimi coinvoilti nella regolazione dell'espressione genica, nella riparazione de DNA e nella risposta allo stress. L'acetilazione gioca un ruolo importante nella trascrizione poiche' responsabile del rimodellamento della cromatina aumentando l'accesso dei fattori di trascrizione al DNA. Una diminuzione dei livelli dell'acetilazione istonica sono associate con un aumento dell'attivita' di trascrizione, mentre un decremento dei livelli di acetilazione e associate con le repression della repressione genica. Infatti nelle cellule tumoralisi osserva frequentemente un'anormale espressione genica. Gli inibitori delle HDAC (HDI) sono potenti induttori dell'arresto della crescita, della differenzione e della morte dellulare per apoptosis in cellule trasformate sia in vitro che in vivo. Ad oggi vi sono sette classi distinte di inibitori delle HDAC e tutti contengono tre elementi strutturali chiave del farmacoforo: a) un gruppo legante lo zinco (R), che coordina gli ioni zinco al fondo della lunga e stretta cavita' del sito attivo; b) un cappuccio o à ¢ capà ¢ (la funzione solfonammidica), che interagisce sia con gli aminoacidi sul margine del sito di legame sia con la superficie della proteina. c) un dominio di legame, il cui ruolo e' assicurare il corretto posizionamento dei gruppi precedenti e di interagire con il canale di legame lipofilo. Seguendo il nostro recente interesse nella ricerca di HDI piu' potenti, ho progettato e sintetizzato una nuova serie di composti contenenti quattro nuove porzioni chelanti lo zinco, quali formilidrazonometilica, 2-formilidrazinocarbonilica, 2,2,2-trifluoro-N-metilencetammidica e 2,2,2-trifluoro-N-metilacetammidica, legate attraverso uno spaziatore ad una solfonammide (Schema 1), responsabile dell'interazione idrofobica con l'HDAC. Studi di docking hanno mostrato che queste molecule si inseriscono perfettamente nella tasca enzimatica dell'HDAC con un ottimo à ¯ Gbind di à ¢ 7,9 kcal/mol che e' una 1 kcal/mol piu' bassa del SAHA, un ottimo inibitore.
The epigenetic control of gene expression is operated through post-translational modifications of chromatin such as acetylation and deacetylation. Two enzymes are responsable for these processes: HAT (histone acetyl transferase) and HDAC (histone deacetylase). HDAC is a group of enzymes involved in regulating gene expression, DNA repair and stress response. Acetylation plays an important role in trascription because it remodels chromatin structure enhancing access to DNA-binding factors. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are associated with repression of gene expression. In tumor cells abnormal gene expression is frequently observed. HDAC inhibitors have been shown to be potent inducers of growth arrest, differentiation, and apoptotic cell death both in vitro and in vivo transformed cells. To date there are seven distinct classes of HDAC inhibitors and all contain three key structural elements to their pharmacophore: a) a zinc-binding group which coordinates the zinc ion at the bottom of the long narrow active site cavity; b) a capping group which interacts both with the amino acids on the rim of the binding cavity and the protein surface; c) a linker domain whose role is to ensure the correct positioning of the two former groups and to in-teract with the lipophilic binding tunnel. Following our recent interest in the research of more potent HDAC inhibitors, we have projected and synthesized a new series of compounds containing four new zinc chelant moieties, i.e. formylhydrazonomethyl, 2-formylhydrazinocarbonyl, 2,2,2-trifluoro-N-methylideneacetamidic and N-(trifluoroacetyl)amidic, linked by an aromatic spacer to an aromatic sulfonamide, that is responsible of hydrophobic interaction with HDAC. Docking studies have shown that these new molecules fit very good in the HDAC enzymatic pocket with a best ï Gbind of â 7,9 kcal/mol that is 1 kcal/mol lower of the corresponding one for SAHA, a good HDAC.

L’épigénétique représente les modifications de l’expression génique, sans altérer la séquence nucléique de l'ADN. L'un des mécanismes de régulation est le remodelage de la chromatine qui s’effectue via les histones acétyltransférases et les histones désacétylases (HDAC) permettant ou non la transcription de gènes. Une expression anormale des HDAC est corrélée à de nombreuses maladies (dépendance à l'alcool, inflammation ainsi que les maladies cardiovasculaires et neurodégénératives, cancers…). Il est primordial de cibler la sélectivité d’une isoforme parmi les 11 connues des HDAC zinc dépendantes pour éviter les effets secondaires. Le but de la recherche était de concevoir et de synthétiser de nouveaux composés, de vérifier leur activité inhibitrice vis-à-vis des HDAC de classe I ou II et leur cytotoxicité sur quatre lignées cellulaires: HaCaT, V79-4, SH-SY5Y et PC12. Ainsi, nous nous sommes concentrés sur les pharmacomodulations du ZBG, de l’espaceur et de la tête de molécules connues tels que le MS-275 (sélectif de la classe I des HDAC), les SAHA et TSA (espaceur en C5 ou C6) avec une forte activité inhibitrice vis-à-vis des HDAC, mais non sélectifs. Nous nous sommes concentrés sur les pharmacomodulations de l'HDACI connu modifiant le domaine de liaison au zinc ZBG (sulfonylhydrazide, catéchol), la nature de l’espaceur (alkyl, aryl) et le groupe de reconnaissance de surface (bis-aryl, adamantyl, indolopyridazinone). Une bibliothèque de 57 nouveaux composés a été créée en trois séries. Aucun d'entre eux n'a montré d'activité inhibitrice satisfaisante. Les composés sélectionnés n'ont pas montré d'activité cytotoxique sur les lignées de cellules neuronales. Sur la base de cette recherche, il est possible de créer de nouveaux composés dans la série indolopyridazinone afin de les tester
Epigenetics represents changes in gene expression without altering the nucleic sequence of DNA. One of the main mechanisms of regulation of gene expression is chromatin remodeling via histone acetyltransferases and histone deacetylases (HDAC), which may or may not allow gene transcription. An abnormal expression of HDACs is correlated with many diseases (alcohol dependence, inflammation as well as cardiovascular and neurodegenerative diseases, cancers...). It is essential to target the selectivity of one isoform among the 11 known zinc-dependent HDACs to avoid side effects. The aim of the research was to design and synthesize new compounds, verify their inhibitory activity against class I or II HDACs and their cytotoxicity on four cell lines: HaCaT, V79-4, SH-SY5Y and PC12. We focused on the pharmacomodulations of ZBG, the linker and the cap of known molecules such as MS-275 (selective for class I of HDACs), SAHA and TSA (spacer in C5 or C6) with a strong inhibitory activity towards HDACs, but not selective. We concentrated on the pharmacomodulations of known HDACI modifying the zinc binding domain (sulfonylhydrazide, catechol), the nature of the spacer (alkyl, aryl) and the surface recognition group (bis-aryl, adamantyl, indolopyridazinone). A library of 57 new compounds was designed in three series. None of them showed satisfactory inhibitory activity. The selected compounds did not show cytotoxic activity on neuronal cell lines. Based on this research, it is possible to create new compounds in the indolopyridazinone series in order to test them

As our understanding of cancer biology increases and novel therapies are developed, an increasing number of predictive biomarkers are becoming clinically available. Aberrant acetylation has been strongly linked to tumourigenesis and the modulation of acetylation through targeting histone deacetylase (HDAC) has led to the introduction of many HDAC inhibitors. To date, two have had regulatory approval for the treatment of cutaneous T cell lymphoma (CTCL). Modifications in chromatin control underpin the mechanism of action of HDAC inhibitors. A genome wide loss-of-function screen identified HR23B as a gene that governs sensitivity to HDAC inhibitors. HR23B shuttles ubiquitinated cargo proteins to the proteasome and elevated levels may contribute to cell death mediated by this pathway. It also governs cell sensitivity to drugs that act directly on the proteasome. HDAC inhibitors influence proteasome activity and there may be a synergistic interaction with proteasome inhibitors. HR23B and HDAC6 interact and HDAC6 may be a negative regulator of apoptosis and a positive regulator of autophagy and through its ability to down-regulate HR23B, may impact on the cellular outcome of HDAC inhibitor treatment. Expression of HR23B has been correlated with clinical response to HDAC inhibitors in a retrospective analysis of CTCL patients. The tissue expression of HR23B and the autophagy marker LC3 has been investigated and there may be a reciprocal relationship in their expression in some tumours which may provide prognostic information and patients with low HR23B expression but high levels of autophagy appear to have a particularly poor prognosis. Well designed, biomarker-driven prospective clinical trials are needed to clarify the predictive and prognostic roles of HR23B.

This diploma thesis deals with the possibility of using HDLC (High-Level Data Link Control) protocol for communication and addressing of smart metering devices with a data concentrator. The HDLC protocol is used in two DLMS/COSEM (Device Language Message Specification/Companion Specification for Energy Metering) communication profiles. To simulate these communication profiles, the most appropriate simulation program is selected. Using this simulator, the first communication profile is implemented and the second one is designed. Communication profile based on TCP/IP (Transmission Control Protocol/Internet Protocol) has been fully implemented. To implement the three-layer HDLC communication profile, all options have been thoroughly explored. Using these findings, a process was designed to guide the full implementation. For the first communication profile the qualitative parameters are measured, which are then plotted and evaluated.

I peptidi sono sempre più impiegati come agenti terapeutici per diverse malattie. Possiedono caratteristiche intermedie tra i farmaci chimici e biologici come dimensioni inferiori, sintetizzabili chimicamente, facilmente modificabili, con bassa tossicità, antigenicità ridotta, affinità di legame e specificità. Queste caratteristiche uniche li rendono una modalità attraente per lo sviluppo di inibitori di interazioni proteina-proteina. Lo scopo del progetto è sviluppare nuovi inibitori a base di peptidi in grado di bloccare l’interazione del fattore di trascrizione “myocyte enhancer 2” (MEF2) con alcuni membri della classe IIa delle istone deacetilasi (HDAC), una sottofamiglia di quattro proteine (HDAC4, HDAC5, HDAC7 e HDAC9) che sono state collegate ad una varietà di tumori solidi ed ematologici. A tal fine sono stati prodotti una serie di peptidi HDAC della classe II impiegando un sistema automatizzato di sintesi peptidica su fase solida, purificati con HPLC su fase inversa e caratterizzati via spettroscopia di massa e dicroismo circolare. L’affinità di legame dei peptidi verso MEF2 è stata testata con saggi di Polarizzazione di Fluorescenza. Infine l’affinità di legame di un peptide è stata incrementata inserendo una struttura sintetica intramolecolare (un braccio), che aiuta a bloccare il peptide in una conformazione specifica, in modo tale da ridurre l’entropia conformazionale.

Histone deacetylase (HDAC) inhibition has recently emerged as a novel therapy for cancer treatment. However, currently approved histone deacetylase inhibitors (HDACi) are pan-inhibitors thus inhibiting all 11 zinc dependent HDAC isoforms including those not involved in tumorigenesis. These inhibitors are also associated with various side effects including a potentially fatal cardiotoxicity. To address these issues, isoform selective HDACi were designed and synthesized. The use of 3-hydroxy-pyridin-2-thione (3HPT) as zinc chelation group resulted in small molecules devoid of HDAC1 inhibition but active against HDAC6 and/or 8. Selected 3HPT containing HDACi displayed anticancer activity against various cancer cell lines including DU145, LNCaP and Jurkat. Surprisingly, the lead-compounds were very potent against Jurkat Jγ cells which are resistant to SAHA-induced apoptosis. HDACi were also targeted to cancer cells using folic or pteroic acids as targeting groups. Incorporation of the folic acid into the HDACi pharmacophoric model resulted in inhibitors selective for HDAC6, whereas pteroic-based HDACi inhibited both HDAC1 and 6. Only the pteroic-based inhibitors displayed anticancer activities against folate receptor overexpressing tumors such KB and HeLa. Furthermore, cell-based studies established the inhibition of HDAC1 as the basis for the anticancer activities of the pteroic-based HDACi.

Orientador: Philippe Remy Bernard Devloo, Sônia Maria Gomes
Tese (doutorado) ¿ Universidade Estadual de Campinas, Instituto de Matemática, Estatística e Computação Científica
Made available in DSpace on 2018-08-19T19:19:39Z (GMT). No. of bitstreams: 1 Siqueira_Denisede_D.pdf: 19845716 bytes, checksum: dbcb91683ea323e390eb0dc4a89e18c3 (MD5) Previous issue date: 2012
Resumo: O estudo do presente trabalho se enquadra na área de Análise Numérica para equações diferenciais utilizando o método de elementos finitos. Especificamente, o objetivo é a construção de espaços de elementos finitos vetoriais Hdiv-conformes. Em formulações mistas de problemas elípticos, que consistem em resolver, simultaneamente, tanto a variável primal p quanto a sua variável dual gradiente de p, espaços do tipo Hdiv são utilizados para aproximar a variável dual. A principal característica de espaços Hdiv-conformes é a continuidade da componente normal nas interfaces dos elementos. Para garantir este comportamento, propõe-se uma sistemática de construção baseada na definição de um campo vetorial ajustado a geometria da partição do domínio, combinada com funções de base escalares H1conformes disponíveis na literatura. Com esta metodologia, são construídas bases hierárquicas, de ordem arbitrária, para subespaços Hdiv-conformes em partições triangulares ou quadrilaterais bidimensionais. No entanto, na simulação de problemas elípticos pela formulação mista, os espaços envolvidos na aproximação das variáveis dual e primal necessitam ser compatíveis para garantir a estabilidade do método. Neste sentido, os espaços desenvolvidos são ajustados de forma a obter taxas ótimas de convergência em um problema de autovalor de Steklov. Considera-se também o acoplamento de formulações clássica e mista para um problema elíptico, no contexto de decomposição de domínios, em que as bases Hdiv-conformes compatibilizadas são aplicadas na formulação mista correspondente
Abstract: The study of this work fits in the area of numerical analysis for differential equations using the finite element method. Specifically, the goal is to construct Hdiv-conformes vectorial finite element spaces. In the mixed formulations of elliptic problems, where the principle is to solve simultaneously both the variable primal p as its dual variable gradient of p, Hdiv spaces are used to approximate the dual variable. The main feature spaces Hdiv-conformes is the continuity of the normal component at the interfaces of elements. To ensure this behavior, we propose a systematic construction based on the definition of a vector field adjusted to the geometry of the domain partition, combined with scalar basis functions H1-conformes available in the literature. With this methodology, hierarchical bases are constructed of arbitrary order, for subspaces Hdiv-conforming considering partitions in two-dimensional triangular or quadrilateral elements. However, the simulation of elliptic problems by the mixed formulation, the spaces involved in the approximation of the primal and dual variables need to be compatible to ensure the stability of the method. In this sense, the approximations spaces are adjusted to obtain optimal rates of convergence for a Steklov eigenvalue problem. It is also consider the coupling of classical and mixed formulations for an elliptical problem in the context of domain decomposition, which the compatibilized bases Hdiv-conformes are applied to the corresponding mixed formulation
Doutorado
Matematica Aplicada
Doutor em Matemática Aplicada

Research performed over the last decade has highlighted the role of HDAC inhibitors (HDACis) as modulators of transcriptional activity and as a potential new class of therapeutic agents against many types of malignacies including breast cancer. These drugs inhibit histone deacetylases, leading to derepression of transcription of various genes that are important for cell cycle arrest and cell death. Trichostatin A (TSA) is one of the best established HDAC inhibitors and has been shown to exhibit potent differentiating and anti-proliferative properties. My data demonstrated that treatment of the MCF-7 breast cancer cell line with TSA causes G2/M phase cell cycle arrest. I characterised the novel F-Box protein called FBXL20 and identified it as a direct target of TSA. This protein is part of a novel E3 ligase complex as it binds Skpl, CUL-I and ROC-I and forms a classical SCF complex that is responsible for ubiquitination and targeting proteins for degradation by the 26S proteasome. I further studied the differences between FBXL20 in human cells and its isoform in rat cells. My data showed that FBXL20 is localised in the cytoplasm, concentrated around the nucleus and plays a role in the TSA-induced effects in MCF-7 cells, through regulating the pro-apoptotic protein Bim. Silencing FBXL20 abolished the G2/M arrest caused by TSA treatment. Although FBXL20 is a similar protein to Skp2 they are regulated by different proteins and exert different functions. These findings provide novel data to demonstrate that known and novel HDACis induce G2/M arrest followed by cell death and that this arrest is dependent on the novel FBXL20 protein in breast cancer cells. Using these newly defined properties of HDACis, I screened a panel of potential HDACis and identified at least one to be more potent than SAHA, which is currently used in the clinical setting and showed that it is able to inhibit proliferation, cause cell cycle arrest and cell death of breast cancer cells.

Le récepteur des oestrogènes (RE) peut moduler l’expression de gènes impliqués dans les processus de prolifération et d’apoptose cellulaires. Cette régulation est possible par le recrutement de complexes corégulateurs. Dans ces complexes, l’activité répressive s’explique essentiellement par la présence d’histones désacétylases (HDAC). Cette famille de protéines est composée de 18 membres classés en 4 groupes. Cette répartition est due aux similarités structurales et de fonctions de ces enzymes. Il y a la classe I (HDAC 1, -2, -3, -8), la classe II (HDAC 4, -5, -6, -7, -9, -10) et la classe IV (HDAC 11) qui ont une activité Zn2+ dépendante alors que la classe III (Sirt1-7) recense les HDAC avec une activité NAD+ dépendante. Des résultats récents du laboratoire ont montré, qu’au niveau ARNm, il y avait un important différentiel d’expression de HDAC9 entre les lignées cellulaires de cancer du sein RE positive et négative ou résistante au tamoxifène. Durant ma thèse, j’ai démontré que la régulation de HDAC9, au niveau de son expression, comme au niveau de ses fonctions, affecte la signalisation oestrogénique en modulant l’expression et l’activité transcriptionnelle de REα. De plus, de nombreuses études ont montré l’activité antiangiogénique d’inhibiteurs de HDAC (HDI) à large spectre comme la TSA (Tricostatin A). La conception et l’identification de HDI, potentiellement sélectifs, comme agents anti-tumoraux et/ou anti-métastatique représente une nouvelle approche de thérapie seule ou combinée avec les produits déjà utilisés dans le traitement du cancer. Ainsi, afin d’identifier et caractériser de nouveaux HDI, j’ai établi un outil bioluminescent pour la détection d’inhibiteurs sélectifs de HDAC. Plusieurs lignées cellulaires Gal4-VP16-HDAC ont été générées dans ce but
The estrogen receptor (ER) can modulate the gene expression with consequences in the cell proliferation, apoptosis. This modulation is possible by the recruitment of coactivator or corepressor complexes. The repression activity is in particular explained by the histones deacetylases (HDACs). This protein family is composed by eighteen members who have been classified in four groups. These HDACs are subdivided on structural and functional similarities. The class I isoforms (HDACs 1, 2, 3 and 8), class II (HDACs 4, 5, 6, 7, 9, 10) and class IV (HDAC11) are Zn-dependent enzymes, whereas class III HDACs (Sirtuins 1-7) are NAD+-dependent. Recent data from the laboratory have shown, at the mRNA level, there is an enormous expression differential of HDAC9 between breast cancer cell line ER positive and negative or OHT resistant cell line. During my thesis, I demonstrated that the regulations of the HDAC9 on the level of its expression as of its role in the various breast cancer cell lines were implicated in the estrogen signaling. This regulation takes place at the transcriptional level and in the ERet#945; activity.In addition, using broad spectrum HDAC inhibitors (HDIs) such as TSA (Tricostatin A), many studies have shown that these inhibitors had antiangiogenic activity. Thus, the design or the identification of selective and potent HDAC inhibitors as agents anti-tumoral and/or anti-metastatic can emerge in a novel opportunity used alone or in combination with the already existing agents for the treatment of cancers. In order to identify and characterize new HDIs, my thesis works consisted to establish bioluminescent cell lines for screening HDAC inhibitors. Different cell GAL4-VP16-HDACs chimeras' models were generated to determine the selectivity of HDIs for the different HDACs

The objective of this work is to analyze the performance of HDLC Balanced Class of Procedures under saturated, full-duplex transmission on error prone links. This thesis extends work done by Bux et al. [8] by considering errors on both the links. Satellite links have long propogation delays compared to terrestrial links, and hence, have longer error recovery times. For such links, errors in acknowledgements have considerable impact on the throughput. In this analysis, the effect of errors in acknowledgements is taken in to consideration. An analytical approach is used to derive performance measures. The concept of "virtual transmission time" introduced by Bux et al is redefined to accommodate the effect of errors in acknowledgements and used in the analysis. Resulting throughput calculations show how various parameters, (e.g. transmission rate, propagation delay, error rate and packet size), interact and determine the performance.
Master of Science

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease requiring a genetic predisposition coupled with an environmental trigger in order for initiation of disease. While the exact pathoaetiology has yet to be determined, both B and T cell dysregulation are thought to contribute to disease. Histone deacetylases (HDACs) are a class of enzymes that hydrolyze the lysine bound acetyl group in both histone and non-histone proteins thereby altering protein structure and function. While the use of pan-HDAC inhibitors has proven to be effective for the treatment of a number of acute diseases, they may not be viable as therapeutics for chronic disease due to cytotoxicity and adverse side effects following long term treatment. We sought to determine whether treatment with a class I and II HDAC inhibitor (HDACi) or a specific HDAC6i would be able to ameliorate disease in lupus-prone NZB/W mice. We found that both the class I and II HDACi (ITF2357) and the HDAC6i (ACY-738) were able to decrease SLE markers of disease including splenomegaly, proteinuria, and anti-dsDNA and IgG production in the sera. Treatment with ITF2357 resulted in an increase in the number of immunosuppressive regulatory T (Treg) cells and a decrease in the pro-inflammatory Th17 phenotype. Furthermore, ITF2357 was found to increase Foxp3 acetylation leading to increased Foxp3 stability allowing for differentiation into the Treg phenotype. ACY-738 treatment was able to correct aberrant bone marrow B cell differentiation while also increasing the number of splenic Treg cells in NZB/W mice. These results suggest that HDAC inhibition is able to ameliorate SLE in NZB/W mice by altering aberrant T and B cell differentiation. Additional studies were performed to further examine the expression and function of different HDAC isoforms in immune cells. Due to the ability of HDAC inhibition to decrease markers of SLE disease as well as alter B and T cell development and differentiation, we sought to determine if specific HDAC isoforms are altered in lupus vs non lupus mice in early and late disease states. We determined the level of class IIb HDAC (HDACs 6, 9, and 10) expression in bone marrow B cells, splenic B and T cells, and glomerular cells from early- and late-disease MRL/lpr lupus-prone mice compared to healthy, age-matched C57BL/6 control mice. Expression of HDAC6 and HDAC9 were significantly increased in all of the tissues tested from MRL/lpr mice. Furthermore, both cytoplasmic and nuclear HDAC activity was increased in diseased MRL/lpr mice, and HDAC activity and expression continued to increase as disease progressed. In vitro treatment with ACY-738, a selective HDAC6i, was able to decrease cytoplasmic HDAC activity and inhibit iNOS production. Furthermore, ACY-738 was able to alter apoptosis through increased Bax expression in B cells. Treatment with ACY-738 was also able to inhibit Hsp90 expression and decrease NF-κB nuclear translocation, which are both upregulated during active SLE. Our studies indicate that HDAC activity contributes to SLE pathogenesis and that the use of isoform-selective HDAC inhibitors may be a viable treatment for SLE.
Ph. D.

Les HDACs humains consistent en une famille de 18 membres différents groupés dans quatre classes. Classe I (HDAC 1-3. 8), classe II (HDAC4-7, 9,10 dont HDAC4, 5, 7 et 9 forment un sous-groupe dû à une organisation structurale commune, alors que HDAC6 est membre de classe IIb), classe III, également désignée sous le nom des sirtuines (SIRT1-7) et classe IV (HDAC11). Les classes I, II et IV partagent des caractéristiques communes, car tous leurs membres sont zinc-dépendants et montrent quelques similitudes de séquence, alors que dans la classe III HDACs il s’agit d’enzymes NAD+-dépendant sans homologie aux autres HDACs. Les pan-inhibiteurs tels que SAHA, qui est actuellement dans des essais cliniques en phase III et a été récemment approuvé pour le traitement du lymphome à cellule T cutané, bloquent les enzymes de classes I et II, alors que le MS275 est un inhibiteur sélectif de la sous-classe I et bloque les activités de HDAC 1, 2 et beaucoup moins efficacement HDAC 3. Des inhibiteurs sélectifs de la classe II ont été également produits, permettant ainsi la dissection des diverses activités de HDACs. Notamment, alors que l'induction de TRAIL semble être associée à l'inhibition des enzymes de la classe I dans les systèmes cancéreux, d'autres fonctions cellulaires dépendent de l'action des HDCAs classe II, comme la régulation de la différentiation, principalement la différentiation cardiaque. HDACis dans des systèmes de différentiation : C2C12 cellules, F9 cellules, cellules 3T3L1. Nous avons examiné MC1568, un nouvel inhibiteur des HDACs, spécifique pour la classe II, dans un modèle leucémique, les cellules U937, et divers systèmes de différentiation, tels que les cellules C2C12 pour la différentiation de muscle, les cellules F9 pour la différentiation endodermale et les cellules 3t3L1 pour la différentiation des adypocytes. De façon intéressante, nous avons constaté que dans les cellules C2C12 l'inhibiteur des HDAC classe 2 bloque l'action catalytique de la classe 2 de HDAC, mais stabilise l'interraction entre le facteur transcriptionnel MEF2D et HDAC4. MEF2D est le principal facteur transcriptionnel, responsable de la différentiation terminale de muscle, et par conséquent l’inhibition de son activité transcriptionnel bloque la différentiation du muscle. D'une manière semblable, dans les deux autres systèmes nous avons évalué l'inhibition de la différentiation et nous avons trouvé que l’activité transcriptionnelle de RAR et PPARγ, essentiels pour la différentiation des deux systèmes respectivement, a été bloqué in vitro dans le premier cas et in vivo dans le deuxième cas. Dans le cas de la différentiation induite par PPARγ, on a utilisé le système des cellules 3T3L1. Le traitement de ces cellules avec Troglitazone induit la différentiation d'adypocytes, alors que la présence du MC1568 le bloque complètement. En analysant le niveau de l'expression de PPARγ pour RT-PCR, nous avons constaté que son niveau a été fortement diminué après traitement avec MC1568. De façon intéressante, le traitement d'une souris exprimant stablement le gène de la luciferase sous le contrôle de l’élément de réponse de PPAR (PPRE-Luc), a bloqué l'activité transcriptionnelle de PPARγ, prouvant que le composé régule l'activité transcriptionnelle de récepteur nucléaire et sa transactivation. Inhibition des HDACs classe II dans des modèles de cancer - Cellules MCF7 Dans des systèmes de cancer, tels que les cellules MCF7 nous avons constaté que l'inhibiteur des HDACs classe 2, MC1568, induisait la sumoylation de HDAC4 ce qui augmente l’activité catalytique de HDAC4 comme cela a été précédemment démontré. La transfection d'un mutant spécifique empêchant la sumoylation de HDAC4 en cellules F9 a diminué la capacité de répression de RAR sur un de ces gènes cibles, le collagène IV. Nous avons démontré que le traitement avec MC1568 régule l'activité transcriptionelle des deux facteurs de transcription, RAR alpha et NF-KB, sur trois gènes cibles, IRF1, TNF et TRAIL. La sumoylation de HDAC4 par RANBP2 réprime transitoirement l'expression de ces gènes cibles. Cette répression est perdue lorsque HDAC4 est dégradé. Leucémie promyelocitaire aigüe (APL), cellules NB4 Les cellules NB4 sont un système cellulaire de leucémie promyelocitaire aigüe (APL). Cette maladie est caractérisée par la présence d'une protéine de fusion, PMLRAR alpha, avec une forte activité de répression due au recrutement augmenté des complexes corépresseurs. Les patients sont soignés avec la thérapie de différentiation par de l'acide rétinoïque (ATRA). Même si le traitement est efficace, il reste toujours les problèmes de résistance et toxicité, causés par les traitements plus longs, qui sont à l’origine du syndrome d'ATRA et des effets tératogènes. Dans les cellules NBA, nous avons constaté que la combinaison d’ATRA et MC1568 agit de manière synergique, induisant une mort indépendante de caspases. L'inhibiteur ZVAD (un pan-inhibiteur pour toutes les caspases) ne bloque pas la mort induite par le MC1568 seul ni par la combinaison des deux composés. La voie des caspases activée a été principalement celle de la caspase 8. En même temps, nous avons trouvé une importante libération du cytochrome C, démontrant la participation de la voie de mort intrinsèque dans la mort induite par ces deux composés. L'utilisation d'un inhibiteur spécifique, le NAC, réduit la mort induite par ATRA et MC1568, prouvant que la voie engagée est celle du NAC. Sachant qu'un inhibiteur de HDAC classe I comme le MS275 induit dans le même système une mort cellulaire caspase-dépendante, principalement via TRAIL, notre observation suggérerait que l'inhibition des HDAC classe 2 active une voie qui est principalement caspase-indépendante. Conclusions Dans mon travail de thèse j'ai évalué l'effet d'un inhibiteur sélectif des HDAC classe II dans plusieurs systèmes et j’ai défini ses effets sur la différenciation et l’apoptose. Cette analyse sera utile pour générer des nouveaux inhibiteurs sélectifs pour les HDACs pour le traitement contre le cancer avec un indice thérapeutique plus élevé que les pan-inhibiteurs des HDACs actuels
Human HDACs comprise a family of 18 different members which are grouped into four classes. Class I (HDAC 1-3,8), class II (HDAC4-7,9,10 of which HDAC4, 5, 7 and 9 form a class II a subgroup due to a common structural organization, while HDAC6 is member of class IIb), class III, also referred to as sirtuins (SIRT1-7) and class IV (HDAC11). Classes I, II and IV HDACs share common features, as all their members are zinc-dependent and exhibit some sequence similarities, while class III HDACs are NAD+-dependent enzymes without homology the other HDACs(1). Pan-inhibitors like SAHA, which is currently in phase III clinical trials and has recently been approved for treatment of cutaneous T cell lymphoma, inhibit both classes I and II enzymes, while MS275 is a subclass I selective inhibitor, which blocks the activities of HDAC 1, 2 and much less efficiently HDAC 3. Also class II selective inhibitors have been generated, marking the onset of a dissection of the various activities of HDACs. Notably, while induction of TRAIL appears to be associated with inhibition of class I enzymes in cancerous systems, other cellular functions involve the action of class II HDACs, like the regulation of the differentiation, mainly the cardiac differentiation. In differentiation systems: C2C12 cells, F9 cells, 3T3L1 cells. We tested a new compound HDAC inhibitor, specific for HDAC class 2 in one leukemic model, U937 cells, and various differentiation systems, such as the C2C12 cells for muscle differentiation, the F9 cells for endodermal differentiation and 3t3l1 cells for adypocyte differentiation. Interestingly we found that in C2C12 cells the HDAC class 2 inhibitor blocked the catalytic action of the HDAC class 2, but stabilized the interaction between the transcriptional factor MEF2D and HDAC4. MEF2D is the main transcriptional factor, responsible for the terminal muscle differentiation, and its transcriptional activity inhibition consequently blocked the muscle differentiation. In a similar way in both the other two systems we evaluated the differentiation inhibition and we found that transcriptional activity of RAR and PPARγ, respectively essential for the differentiation of the two systems, was blocked either in vitro in the first case either in vivo in the second case. In the case of the PPARγ induced differentiation, the experiments were assayed in the 3T3L1 cells. The treatment of these cells with Troglitazone induced adypocyte differentiation, while the presence of the MC1568 blocked it completely. Analyzing the Pparg’s expression level for RT-PCR, we found that its level was highly decreased after treatment with the MC1568. Interestingly the treatment of a mouse stably expressing a PPRE-luc, PPAR responsive elements luciferase reporter, blocked the transcriptional activity of PPARγ, showing in this case that the compound was regulating both the nuclear receptor’s transcriptional activity and the transactivation. In tumour model: MCF7 cells In cancer systems, such as MCF7 cells we found that the HDAC class 2 inhibitor MC1568 was inducing HDAC4 sumoylation, that, as previously showed (2) increases the HDAC4 catalytic action. The transfection of a specific HDAC4 sumoylation mutant in F9 cells decreased the repressory power on a note RAR target gene, Collagen IV. We were able to demonstrate that the treatment with MC1568 was regulating the activity of two transcriptional factor, RAR alpha and NF-KB, on three target genes, IRF1, TNF and TRAIL. The HDAC4 sumoylation mediated by RANBP2 repressed transiently the expression of these target genes, that were up-regulated after the complete degradation of HDAC4. Acute promyelocitic leukemia (APL), NB4 cells The NB4 cells is a cellular system of acute promyelocitic leukemia (APL). This disease is characterized by the presence of a fusion protein, PMLRAR alpha, with a strong repressory activity, mediated by the enhanced recruitment of corepressory complexes. The patients are treated with the differentiation therapy with retinoic acid (ATRA). Even if it is quite efficient to treat it, it still remains the problem of resistances, recidives and toxicity, done by the longer treatments, that give the, as said, ATRA syndrome and teratogenicity. In these cells we found that the combination among ATRA and MC1568 seems synergic, inducing a caspase independent cell death, even if in presence of caspases activation. The ZVAD inhibitor (pan-inhibitor for all caspases) failed to block the death induced by the MC1568 and by the combination among the two compounds. Analyzing the activation of the caspases it seemed that the pathway activated was mainly mediated by caspase 8. At the same time we found a strong release of cytochrome C, demonstrating the involvement of the mitochondria in the death induced by these two compounds. The use of a specific inhibitor, the NAC inhibitor, was able to reduce the death mediated by ATRA and MC1568, showing that the pathway was mainly regulated by the NAC dependent way. At this time, we are arrived to select the pathway regulated by this compound. Knowing that an HDAC inhibitor like MS275, specific inhibitor for class 1 HDACs, induce in the same system a caspase-dependent cell death, mainly regulated by TRAIL3, our observation would suggest that the class 2 HDAC inhibition is specifically regulating a pathway that is mainly caspase-independent. This observation could be usefull for treatment therapy, not only ammeliorating the treatment with retinoids, but overcoming possible resistances mediated by mutations and alteration in receptor expression in tumoral cells. Conclusions In my work I have evaluated the effect of a class 2 HDAC inhibitor in several systems, differentiative and cancerous, highlighting its effects in transcriptional regulation and cell death
Nell’uomo gli enzimi de acetilanti gli istoni appartengono ad una famiglia di 18 membri che sono raggruppati in 4 classi. La classe I (HDAC1-3,8), classe II (HDAC4-7,9,10 di cui HDAC4, 5,7,9 formano un subgruppo dovuta ad una comune organizzazione strutturale, mentre HDAC6 è un membro della classe 2), classe III, denominata come sirtuine (SIRT1-7) e classe IV (HDAC11). Le HDAC appartenenti alle classi I, II, IV sono comunemente dipendenti dallo zinco, mentre le HDAC appartenenti alla classe III sono enzimi dipendenti dal NAD +(1). SAHA, un pan inibitore delle HDAC, inibisce entrambi le classi I e II. Attualmente è in phase III clinical trials ed è stato recentemente approvato per il trattamento del linfoma cutaneo delle cellule T. MS275, invece, è un inibitore selettivo per la subclasse I, bloccando preferenzialmente HDAC1,2. Recentemente sono stati sintetizzati anche inibitori selettivi per la classe II, rendendo possibile lo studio delle varie funzioni delle differenti classi delle HDAC. Come è noto, mentre l’induzione di TRAIL sembra essere associata con l’inibizione di enzimi classe I in sistemi cancerosi, le HDAC di classe II sembrano essere coinvolte in altra funzioni cellulari, come la regolazione del differenziamento, principalmente quello cardiaco. Inibitori delle HDAC in sistemi differenziativi: le cellule C2C12, le cellule F9 e le cellule 3T3L1. Abbiamo testato un nuovo composto inibitore HDAC, specifico per la classe 2 in un modello leucemico, le cellule U937, e vari sistemi differenziativi, come le cellule C2C12 per il differenziamento cardiaco, le cellule F9 per il differenziamento endodermico e le cellule 3T3L1 per il differenziamento adipocitario. Abbiamo scoperto che nelle cellule C2C12 l’inibitore HDAC classe 2 stabilizzò l’interazione tra il fattore trascrizionale MEF2D ed HDAC4, pur bloccando l’azione catalitica delle HDAC classe II. MEF2D è il principale fattore trascrizionale resposabile del differenziamento cardiaco terminale e l’inibizione della sua attività trascrizionale conseguentemente bloccò il differenziamento muscolare. Negli altri due sistemi, similmente, noi abbiamo riscontrato l’inibizione del differenziamento ed abbiamo trovato che l’attività trascrizionale di RAR e PPARγ, essenziali per il differenziamento dei relativi sistemi differenziativi, fu bloccata sia in vitro nel primo caso che anche in vivo nel secondo. Nel caso del sistema differenziativo indotto da PPARγ, gli esperimenti furono fatti nelle 3T3L1. Il trattamento di queste cellule con Troglitazione indusse il differenziamento adipocitario, mentre la presenza dell’MC1568 lo bloccò completamente. Analizzando il livello di espressione di PPARγ per RT-PCR, abbiamo ritrovato che il suo livello era altamente decrementato dopo trattamento con MC1568. Abbiamo notato inoltre che il trattamento di un topo transgenico esprimente stabilmente il PPRE-luc, reporter luciferasi degli elementi responsivi a PPAR, bloccò completamente l’attività trascrizionale di PPARγ, mostrando in questo caso che il composto regolava sia l’attività trascrizionale del nuclear receptor sia il suo livello di espressione. In modelli cancerosi: le cellule MCF7 In sistemi cancerosi, così come le cellule MCF7 abbiamo riscontrato che lo specifico inibitore delle HDAC di classe II induceva la sumoilazione di HDAC4, che, come previamente mostrato (2) incrementa l’azione catalitica di HDAC4. La trasfezione di un mutante di HDAC4 specifico per il sito di sumoilazione nelle cellule F9 decrementò il potere repressorio su un noto target di RAR, CollagenIV. Abbiamo dimostrato che il trattamento con MC1568 regolò l’attività di due fattori trascrizionali, quali RAR alpha ed NF-KB, su tre geni target, IFR1, TNF e TRAIL. La sumoilazione di HDAC4 mediata da RANBP2 represse in modo transiente l’espressione di questi geni target, che furono up-regolati dopo la completa degradazione di HDAC4. Leucemia promielocitica acuta (APL), NB4 cells Le cellule NB4 è un sistema cellulare di leucemia promielocitica acuta (APL). Questa malattia è caratterizzata dalla presenza di una proteina di fusione, PMLRAR alpha, con un forte attività repressoria. I pazienti sono attualmente curati con la terapia differenziativa con acido retinoico (ATRA). Anche se è un trattamento molto efficiente, vi sono ancora casi di recidività, resistenze e tossicità al trattamento, dovuto principalmente ai trattamenti prolungati, dando luogo alla cosiddetta ATRA sindrome e teratogenicità. In queste cellule abbiamo scoperto il sinergismo tra ATRA e MC1568. La combinazione delle due droghe ha indotto una morte caspasi-indipendente, anche se in presenza di attivazione delle caspasi. L’inibitore ZVAD, infatti, fallì a bloccare la morte indotta dall’MC1568 e dalla combinazione tra i due composti. Analizzando l’attivazione delle caspasi, abbiamo riscontrato che il pathway maggiormente attivato è quello della caspasi 8. Allo stesso tempo abbiamo ritrovato un forte rilascio del citocromo C, dimostrando il coinvolgimento del mitocondrio nella morte indotta da questi due composti. L’uso di un inibitore specifico dei reattivi dell’ossigeno, N-acetyl-cisteina, ridusse significativamente la morte indotta, definendo il ruolo chiave dei ROS. Sapendo che un inibitore delle HDAC quale MS275, un inibitore specifico delle HDAC di classe 1, inducesse nello stesso sistema una morte caspasi dipendente, principalmente regolata da TRAIL3, la nostra osservazione suggerirebbe che l’inibizione specifica delle HDAC di classe II regola specificamente un pathway caspasi-indipendente, utile per migliorare il trattamento con retinoidi, ma soprattutto per curare quei pazienti con resistenze dovute a mutazioni o alterazione nella espressione del recettore in cellule tumorali. In Conclusione Nel mio lavoro ho studiato l’effetto di un inibitore specifico delle HDAC di classe II in svariati sistemi, differenziativi e cancerosi, concentrandomi sugli effetti nella regolazione trascrizionale e la morte cellulare

Asthma is chronic inflammatory disease of the lower airways that is both, genetically inherited and environmentally influenced. This project investigated how molecular mechanisms known to be influenced both genetically and environmentally, contribute to the onset of asthma.

The tumor suppressor gene RB regulates cell proliferation at the G1/S transition of the cell cycle. The retinoblastoma protein pRB associates with both HDAC-dependent and independent mechanisms to actively repress E2F-dependent genes required for entry into S phase. The retinoblastoma binding protein 1 RBP1 recruits the mSin3A/HDAC1 co-repressor complex to the pocket of pRB at growth arrest and accounts for the majority of the HDAC activity associated with pRB. However, transcriptional repression by RBP1 also involves HDAC-independent activities because repression is only partially relieved by the HDAC inhibitor Trichostatin A. This activity is mediated in part by residues 241 to 452 of RBP1 designated as the R1 domain. Hypermapping studies on the previously defined R1 domain of RBP1 revealed that amino acids 400 to 452 of RB1 are sufficient to mediate HDAC-independent repression. Inspection of the minimal R1 region located two copies of the SUMO consensus motif Psi-K-X-E and subsequent experiments demonstrated that the R1 domain is post-translationally modified by SUMO on lysine 418 and 444. In addition, transcriptional repression by the R1 domain was abrogated by either mutagenesis of both SUMO acceptor lysines or in the presence of a SUMO specific protease implying that SUMO modification modulates HDAC-independent transcriptional repression by RBP1.

Malaria is a leading cause of morbidity and mortality, causing more than 400,000 deaths per year. Malaria is caused by parasites of the Plasmodium genus with most deaths due to P. falciparum infection. The control of malaria is complicated by the lack of a widely effective vaccine, the spread of mosquito resistance to insecticides and Plasmodium parasite resistance to available drugs, including the gold standard artemisinin-combination therapies. Thus, there is an urgent requirement for the development of new antimalarials, in particular those with different modes of action to existing drugs to limit potential problems of cross-resistance. Plasmodium species have a complex lifecycle that includes transmission from the female Anopheles mosquito vector to a human host requiring significant morphological changes. These morphological changes are associated with stage-specific changes in transcription regulated by epigenetic mechanisms. The proteins involved in these processes are potential new therapeutic targets for malaria. This includes histone deacetylases (HDACs), which together with histone acetyltransferases (HATs), are involved in reversible posttranslational acetylation of histone and non-histone proteins, regulating transcription and other cellular processes. To date, over 650 HDAC inhibitors have been investigated for in vitro activity against malaria parasites. Some inhibitors, particularly those with a hydroxamic acid zinc-binding group that targets inhibitors to the HDAC active site, have demonstrated low nM in vitro potency against P. falciparum and selectivity for the parasite over human cells. However, antiplasmodial HDAC inhibitor drug development has been hindered by factors including the lack of recombinant P. falciparum HDACs (only one available and purity is low), the lack of HDAC crystal structures (none available) and low throughput activity assays that are largely indirect measures of HDAC inhibition. Without these tools, mode of action studies, the rational design of new and improved inhibitors and the prioritisation of compounds for preclinical testing remains difficult. To address some of these challenges and further progress the development of antimalarial HDAC inhibitors, the current study employed a multi-pronged approach, including: (i) investigating the in vitro and in vivo activity of new HDAC inhibitors; (ii) establishing a higher throughput ELISA method to analyse P. falciparum lysine acetylation alterations and; (iii) developing a quantitative structure-activity relationship (QSAR) model based on classification algorithms. HDAC inhibitors typically have a pharmacophore comprising a zinc-binding group that interacts with the zinc ion in the active site of the enzyme, a linker unit and a cap group promoting hydrophobic interaction with amino acid residues at the entry of the active site. Here, a set of 26 new HDAC inhibitors with a peptoid-based scaffold was tested in vitro against drug sensitive asexual intraerythrocytic-stage P. falciparum 3D7 parasites. The set are analogues of compounds that have previously shown in vitro dual-stage antiplasmodial activity against asexual intraerythrocytic and exoerythrocytic stages and includes 16 compounds with a hydroxamic acid zinc-binding group and 10 prodrugs of this compound class. The unprotected hydroxamate-based inhibitors demonstrated growth inhibition of P. falciparum 3D7 asexual intraerythrocytic-stage parasites in the nanomolar to micromolar range (50% growth inhibition values (PfIC50) 0.008-1.04 μM) and up to 1,250-fold selectivity (selectivity indices (SI; PfIC50/human cell IC50): 10-1,250) for the parasite compared to human cells. Structure-activity relationship (SAR) analysis of cap region residues (carbonyl region, carboxylic region and isocyanide region) indicated that benzyl groups in the isocyanide region and alkyl groups in the para position of the carboxylic region are associated with increased antiplasmodial activity. In addition, methyl groups in the carbonyl region of the cap group demonstrated reduced cytotoxicity against neonatal foreskin fibroblasts (NFF), however, also somewhat reduced activity against asexual blood-stage parasites. Work by collaborators demonstrated micromolar in vitro activity of several compounds of this set against exoerythrocytic P. berghei parasite forms indicating dual-stage activity. The compound with the greatest dual-stage activity displayed an IC50 of 8 nM against asexual blood-stage P. falciparum and an IC50 of 60 nM against exoerythrocytic P. berghei in vitro. Compounds with PfIC50 of 100 nM or lower were tested against the multi-drug resistant P. falciparum Dd2 line (resistant to chloroquine, pyrimethamine, mefloquine, and other antimalarial drugs), and demonstrated a resistance index (RI) <1 indicating a lack of cross-resistance by this parasite line. The same subset of compounds was investigated for their ability to hyperacetylate P. falciparum histone H4; differential effects were observed with some compounds causing up to ~2.5-fold hyperacetylation compared to untreated controls. 10 prodrug peptoid-based HDAC inhibitors were also investigated. The prodrug strategy seeks to make the hydroxamic acid-based inhibitors more stable and bioavailable for in vivo applications as they are prone to degradation processes such as hydrolysis or reduction. These compounds were synthesised with masked hydroxamate functionalities that may undergo activation in vitro. Preliminary data demonstrated in vitro PfIC50 of 0.014-1.75 μM and 6-642-fold selectivity for the parasite over human fibroblasts. Three of these compounds displayed PfIC50 <0.1 μM and SI >100 and may therefore be of interest in further studies. Based on the in vitro antiplasmodial activity, selectivity and chemical diversity in the cap region, five peptoid-based compounds (3a, 3c, 3f, 3m, 3n, Pf3D7 IC50 0.008-0.034 μM, SI 97-625) were further investigated for in vivo efficacy against Plasmodium parasites. In addition, four analogues of the tethered phenylbutyrate-based HDAC inhibitor AR42 (Pf3D7 IC50 0.02 μM, SI 39) were also investigated in vivo (JT21b, JT83, JT92a, JT94; Pf3D7 IC50 0.005-0.21 μM, SI 55-118, (data generated by Dr MJ Chua, personal communication)). AR42 is currently in phase 1 clinical trials against various types of cancer and demonstrates an improved pharmacokinetic profile compared to a number of clinically approved HDAC inhibitors (e.g. AR42 Cmax 14.7 μM compared to vorinostat Cmax 1.9 μM, AR42 t1/2 11.1 h compared to vorinostat t1/2 0.75 h; tested in mice). AR42 analogues were of interest as AR42 has previously been shown to cure Plasmodium infections in mice (Dr MJ Chua, Griffith Institute for Drug Discovery; unpublished). While the two analogue sets differ significantly in linker and cap group, both bear a hydroxamic acid zinc-binding group. Compounds were tested in groups of two female BALB/c mice infected with P. berghei ANKA infected erythrocytes. Dosing was via oral gavage at 25 mg/kg twice daily with four hours between dosing (beginning 2 h post infection) for four consecutive days. Peripheral blood parasitemia was monitored by microscopic evaluation of stained thin blood films from day four post infection. None of the peptoid-based HDAC inhibitors attenuated P. berghei growth in BALB/c mice by more than 33% (3f (31%) and 3n (33%) on day 6 post infection). Data from collaborators demonstrated 3n to have the best metabolic stability (t1/2 271 min, Clint 6 μL/min/mg in mice; Prof Finn Hansen, University of Bonn, Germany) which may have contributed to this compound’s improved activity compared to some other analogues. In comparison, AR42 and two if its analogues cured mice of infection (AR42, 1 of 2 mice; JT21b, 2 of 2 mice; JT83 2 of 2 mice), up until day 24 post infection, at which point the mice were euthanised. AR42 and analogues are the first demonstration of oral cures in mice with a HDAC inhibitor (manuscript in preparation) and these data will be pursued in future work to further develop this HDAC inhibitor chemotype for malaria. One of the current limitations in the field is the lack of recombinant P. falciparum HDACs and the need to rely on low throughput assays to demonstrate HDAC inhibitor action via reduced total deacetylase activity or in situ lysine acetylation alterations. While deacetylase assays do not allow the differentiation of compound effects, Western blot using different acetyl-lysine antibodies can reveal compound specific acetylation profiles. Here, two higher throughput methods, dot blot and ELISA, were investigated to assess the effects of HDAC inhibitors on lysine acetylation. Using the control hydroxamate HDAC inhibitor vorinostat (first HDAC inhibitor clinically approved for cancer), the ELISA method was demonstrated to be more reliable than dot blot in detecting acetylation changes in protein lysates from P. falciparum trophozoites exposed to compound for 3 h. ELISA was therefore used to investigate histone H3 and H4 lysine acetylation alterations following exposure of P. falciparum to six commercially available anti-cancer HDAC inhibitors (vorinostat, panobinostat, trichostatin A, romidepsin, entinostat and tubastatin A). All compounds have in vitro activity against asexual intraerythrocytic P. falciparum parasites (Pf3D7), with tubastatin A activity reported for the first time here (PfIC50 0.15 ± 0.03 μM). All compounds were also shown to inhibit >84% deacetylase activity using P. falciparum protein lysates in an in vitro assay at 1 μM, with the exception of entinostat (~50% inhibition at 1 μM); this compound was also the least active against the parasite (PfIC50 11.5 μM). Using ELISA, vorinostat, panobinostat, trichostatin A, romidepsin and entinostat were all found to cause a ~3-fold increase in the signal detected using an anti-tetra-acetyl-lysine antibody. In comparison, the only human HDAC6-specific inhibitor tested, tubastatin A, caused 1.8-fold histone H4 hyperacetylation compared to the control. Further investigations of the individual N-terminal H4 lysine residues using antibodies specific to acetylated lysine 5, 8, 12 or 16 revealed that all compounds, except tubastatin A, caused hyperacetylation using each antibody. No differential effect was observed for histone H3 acetylation, with all compounds causing an ~1.8-fold increased signal using an acetyl-H3 antibody. The new ELISA method developed here provides a higher throughput way to assess differential compound induced lysine acetylation alterations in P. falciparum and therefore represents a valuable new tool to aid the investigation of HDAC inhibitors for malaria. As discussed above, the lack of tools, such as recombinant P. falciparum HDAC proteins, crystal structures and homology models, has meant that the identification of antiplasmodial HDAC inhibitors has been limited to whole-cell screening approaches which can be time-consuming and costly. To begin to address this problem, quantitative structure-activity relationship (QSAR) models were developed based on logistic algorithms with the aim of providing a new tool to triage compounds for in vitro testing. A database of 457 antiplasmodial HDAC inhibitors was assembled with published data on PfIC50 and, for 292 of those compounds with data on plasmodial selectivity. Two independent prediction algorithms based on logistic regression were developed to classify (1) antiplasmodial activity or (2) selectivity of hydroxamate-based HDAC inhibitors. Seven different activity and five different selectivity models were built, each with individual decision cut-offs defining active/selective and non-active/unselective compounds (e.g. PfIC50: active compound <0.1 μM> non-active compound; SI: selective compound >100< unselective compound). Activity model A7 revealed the highest prediction performance by predicting 93% of the training compound set and 87% of the external test compound set correctly. Cross validation revealed a prediction accuracy of 91%. The most accurate selectivity model S4 demonstrated a slightly poorer prediction performance due to a much smaller initial data set as not all the HDAC inhibitors had reported selectivity information (64%). Despite this, the selectivity model demonstrated an internal prediction accuracy of 91%, a cross-validated (internal) prediction accuracy of 82% and an external prediction accuracy of moderate 72%. To validate the prediction performance of the activity model further, they were applied to a set of 22 experimentally untested compounds (validation set) and the prediction performance compared to their experimental antiplasmodial activity. Applying prediction model A7 to this compound set predicted three hit compounds (two of which were confirmed by experimental assay data) and 12 non-actives (confirmed for 11 based on experimental assay data). The experimental PfIC50 assessment revealed asexual blood-stage PfIC50s for the whole set in the nanomolar to micromolar range (PfIC50 0.006-8.45 μM; data from Dr MJ Chua), with the correctly predicted hits (S2_E10 and LD016) having PfIC50 <0.008 μM. Overall, virtual screen using QSAR model A7 identified 87% of the validation compounds correctly and revealed high prediction specificity, identifying 92% of the non-active compounds correctly. Due to a lack of available data sets with selectivity index information (and time constraints for this project), the selectivity models were not able to be tested with an external set. These activity and selectivity QSAR models are the first generated for antiplasmodial HDAC inhibitors. These models will aid the in silico assessment of antiplasmodial activity and selectivity of hydroxamate-based HDAC inhibitors and therefore represent useful new tools in the investigation of HDAC inhibitors for malaria. In summary, data presented in this thesis include the identification of novel antiplasmodial HDAC inhibitors with activity against asexual intraerythrocytic-stage P. falciparum parasites, in vivo data demonstrating oral cures in mice for two analogues of the anti-cancer HDAC inhibitor AR42, a new ELISA method to allow higher throughput assessment of HDAC inhibitor induced changes to histone lysine residues and the first antiplasmodial HDAC inhibitor QSAR models. HDAC inhibitors identified in this study with promising in vitro and in vivo antiplasmodial activity profiles are new starting points for further development of HDAC inhibitors for malaria. In addition, the in vitro and in silico approaches developed in this study are useful new tools to facilitate the discovery of HDAC inhibitors and the understanding of their biological effects on the parasite.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Histone deacetylases (HDACs) are enzymes involved in the remodeling of chromatin. In recent years, inhibition of HDACs has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer. In fact, HDAC inhibitors (HDACi) promote apoptosis, induce cell cycle arrest and differentiation of tumor cells, by mechanisms which remain in part unknown. T-cell acute lymphoblastic leukemia (T-ALL) is a pediatric malignancy characterized by clonal expansion of lymphoid progenitors. Although the majority of pediatric T-ALL patients can be cured by current protocols, about one fourth of patients has chemotherapy-resistant disease or relapse after therapy and novel therapeutic approaches are required. In our study, we analyzed the effects of HDACi on seven transcription factors important in T-ALL pathogenesis (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) using both established T-ALL cell lines and patient-derived T-ALL xenografts previously obtained in our laboratory. In particular, we focused on transcription factors that define specific T-ALL subgroups (TAL/LMO, TLX1, TLX3) and we included members of the Notch family (NOTCH1 and NOTCH3) and c-MYB in view of their transversal role in T-ALL. In vitro analysis highlighted transcriptional down-regulation of C-MYB and TAL1, a post-translation regulation of NOTCH1 and NOTCH3 and the regulation of the transcriptional activity of TLX1 and TLX3 following HDAC inhibition. These biochemical effects were linked to increased apoptosis and impaired proliferation both in T-ALL cell lines and patients-derived cells, partially dependent on NOTCH1 and NOTCH3. We next investigated the in vivo effects of an HDACi in T-ALL xenografts belonging to specific T-ALL subgroups. Interestingly, PD-TALL8 (TLX1) and PD-TALL16 (TLX3) had better response to treatment compared to PD-TALL12 and PD-TALL9 (TAL/LMO). In fact, the HDACi dramatically decreased leukemic cells infiltrating the spleen and the bone marrow in TLX-driven xenografts, whereas this drug had modest or minimal effects on TAL/LMO xenografts. Taken together, these results identify TLX1 and TLX3 T-ALL subgroups as potential candidates for therapeutic treatment with HDACi.
Le Istone Deacetilasi (HDACs) sono enzimi coinvolti nel rimodellamento della cromatina. Negli ultimi anni è emerso come l’inibizione delle HDACs potrebbe essere utilizzata come strategia per ripristinare l’alterata regolazione epigenetica che si riscontra nei tumori. Infatti, gli inibitori delle HDAC (HDACi) inducono apoptosi, arresto del ciclo cellulare e differenziamento delle cellule tumorali, ma i meccanismi molecolari alla base di questi fenomeni rimangono poco chiari. La leucemia linfoblastica acuta a cellule T (T-ALL) è un tumore pediatrico caratterizzato dall’espansione clonale di progenitori linfoidi. Nonostante la maggioranza dei pazienti pediatrici affetti da T-ALL siano curati in modo efficace utilizzando gli attuali protocolli terapeutici, circa un quarto dei pazienti manifesta resistenza alla terapia o presenta ricadute e dunque emerge la necessità di nuovi approcci terapeutici. In questo studio abbiamo analizzato gli effetti degli HDACi nei confronti di sette fattori di trascrizione implicati nella patogenesi della T-ALL (NOTCH1, NOTCH3, c-MYB, TAL1, TLX1, TLX3 and LMO2) utilizzando sia linee cellulari stabilizzate, sia modelli murini di T-ALL precedentemente sviluppati nel nostro laboratorio a partire da cellule di pazienti. In particolare, ci siamo concentrati su fattori trascrizionali che identificano specifici sottogruppi di T-ALL (TAL/LMO, TLX1 e TLX3) e abbiamo incluso nell’analisi due membri della famiglia dei recettori Notch (NOTCH1 and NOTCH3) e c-MYB in virtù del loro ruolo oncogenico in questa patologia. Le analisi in vitro hanno evidenziato diversi meccanismi di regolazione dei vari fattori da parte degli HDACi. TAL1 e c-MYB risultano regolati a livello trascrizionale, NOTCH1 e NOTCH3 presentano una regolazione post-traduzionale e, nel caso di TLX 1 e TLX3, è presente una regolazione diretta della loro capacità trascrizionale. Gli effetti a livello di proteina si legano all’induzione di apoptosi e all’inibizione della proliferazione sia nelle linee cellulari, sia nelle cellule derivate da paziente e risultano essere parzialmente dovute alla down-modulazione di NOTCH1 e NOTCH3. In seguito siamo andati ad indagare la risposta in vivo di un HDACi in xenografts di T-ALL appartenenti a specifici sottogruppi genetici. E’ interessante notare che il trattamento ha avuto il maggiore risultato nelle PD-TALL8 (TLX1) e nelle PD-TALL16 (TLX3) rispetto alle PD-TALL12 e le PD-TALL9 (entrambe TAL/LMO). Infatti, il trattamento con HDACi negli xenografts di tipo TLX determina una riduzione dell’infiltrazione da parte delle cellule leucemiche nella milza e nel midollo mentre gli effetti ottenuti negli xenografts TAL/LMO risultano modesti o addirittura nulli. In conclusione, i dati ottenuti identificano i pazienti di T-ALL appartenenti ai sottogruppi TLX1 e TLX3 come potenziali candidati per il trattamento a scopo terapeutico con HDACi.

En réponse à des stress environnementaux, la cellule met en place une réponse rapide et transitoire visant à assurer sa survie. Cette réponse se traduit par l'activation du facteur HSF1(Heat shock factor 1) qui induit l'expression des gènes codant pour des protéines de choc thermique (ou HSP). L'activation des gènes hsp s'accompagne de la répression de la plupart des autres gènes cellulaires. Si l'on connait assez bien aujourd'hui les mécanismes associés à l'activation des gènes de choc thermique, peu de données existent concernant les mécanismes mis en jeu dans l'inactivation globale. Nous avons engagé un travail visant à caractériser les modifications épigénétiques qui accompagnent cette répression, ainsi qu'à identifier les acteurs impliqués. Par des approches moléculaires et in situ nous avons montré que les HDACs (Histones Décaétylases) sont de nouveaux régulateurs de la réponse au stress. Le stress thermique induit une régulation fine de l'épigénome, notamment une déacétylation globale des histones de cœur, médiée par des HDACS de classe l, HDAC1 et 2. Au niveau du cytoplasme, les HDACs régulent également la réponse au stress. En effet, lors d'un stress protéotoxique, nous avons montré qu'HDAC6 joue un rôle indispensable dans l'initiation de cette réponse, en dissociant le facteur HSFl de ses régulateurs négatifs. En conséquence, HDAC6 joue un rôle dans l'induction des protéines HSPs en réponse à ce stress de type agrégats. En conclusion, en identifiant les HDACs comme de nouveaux facteurs de la réponse au stress, nos travaux permettent de mettre en lien entre deux cibles faisant l'objet de nombreux travaux en cancérologie: HSPs et HDACs
Ln response to environmental stress (heat shock, hypoxia, heavy metals exposure), cells have developed rapid and transitory mechanisms to protect themselves from the stress-induced damages. This stress response is characterized by the activation of HSF1 (Heat Shock Factor1), a key factor involved in the induction of the HSP (Heat Shock Proteins) encoded genes. Ln contrast toheat shock genes induction, most of the genome is repressed du ring stress. If the mechanisms involved in the activation of HSP genes have been investigated in details, less is known about the global repression of the genome. We started to investigate the epigenetic mechanisms that underline this genome repression and identify the molecular basis of this phenomenon. By molecular and in situ approaches, we showed that HDACs (Histone Deacetylases) are new regulators of stress response. Heat shock induces major epigenetic changes, specially a global deacetylation of core histones. We showed that class 1 HDAC, HDACl and HDAC2 mediates the heat-induced deacetylation. This event is regulated by HSF1, probably through its interaction with HDACl and HDAC2. Ln the cytoplasm, HDACS are also able to regulate stress response. Indeed, upon proteotoxic stress for example, proteasome inhibition, we showed that HDAC6 play a critical role in initiating the stress response. It mediates the dissociation of HSFl from its repressor complex and HDAC6 has an impact in HSP induction in response to stress. Ln conclusion, we identify HDACs as new important factors of stress response. Thanks to this work, we have linked two classes of proteins that are targeted by anti-cancer therapy: HSPs and HDACs

Le déséquilibre HAT/HDAC aurait une influence sur le développement de certains cancers ainsi que dans l’addiction à l’alcool ou à la cocaïne. En inhibant les histones désacétylases, le taux d'acétylation de la chromatine augmente ce qui permet l’accès aux facteurs de transcription et l'expression des gènes. Aujourd'hui, il existe de nombreux inhibiteurs d'HDAC de structures diverses, mais ils ne sont pas spécifiques et présentent des effets secondaires importants. Les inhibiteurs d'HDAC comme le butyrate de sodium ou le MS-275 ont montré une modification de la dépendance à l'alcool chez le rat. MS-275 inhibe principalement la classe I de HDAC et conformément à ces observations nous nous intéressons aux inhibiteurs les plus sélectifs de la classe I tels que le Largazole thiol et le RedFK228. Notre but est de synthétiser de nouveaux cyclodepsipeptides analogues afin d'obtenir un inhibiteur sélectif de la classe I. Les HDAC de la classe I sont Zn-dépendants, ces analogues auront un groupement sulfonylhydrazide ayant une bonne affinité pour l’ion Zn2+ (ZBG). Il sera relié au cyclodepsipeptide par un bras espaceur dont la longueur sera adaptée (n = 2, 3). Une autre pharmacomodulation concerne l'incorporation d’hétérocycles différents (oxazole, thiazole et pyridine). Les inhibitions de ces composés ont pu être testées sur HDAC1, HDAC3 et HDAC6. Un composé a une spécificité pour HDAC3 et un autre a une spécificité pour HDAC1. Les tests sur des rats "binger" permettent de penser que HDAC1 est impliqué dans ce model de consommation et non HDAC3
The imbalance HAT/HDAC would influence the development of cancers and alcohol or cocaine addiction. HDAC inhibition allows increase of both acetylation rate and gene expression. Today, there are many structurally diverse potent, but non-specific HDAC inhibitors displaying important side-effects. HDAC inhibitors such as sodium butyrate or MS-275 have been shown to alter the alcohol dependence in the rat. MS-275 inhibits mainly class I of HDAC and in line with these observations we are interested in more selective class I inhibitors such as Largazole thiol and RedFK228. Our purpose is to synthesize new cyclodepsipeptides analogues in order to obtain selective class I inhibitor. HDAC class I is a Zn-dependent enzyme and our target molecules have sulfonylhydrazide function as efficient Zinc binding group (ZBG). Additional pharmacomodulations concern the incorporation of different heterocycles (oxazole, thiazole, pyridine) and varying linker lengths (n = 2, 3). Inhibitions of these compounds have been tested on HDAC1, HDAC3 and HDAC6. A compound has specificity for HDAC3 and another has specificity for HDAC1. Tests on rats "binger" suggest that HDAC1 is involved in this model of consumption and not HDAC3

NAD+-Dependent histone deacetylases, also called sirtuins, are a family of enzymes that catalyse the cleavage of acetyl groups from lysine amino acid residues in histones and a variety of nonhistone proteins, playing a critical role in numerous biological processes. They have been found to be deregulated in several age-related diseases such as cancer and neurological disorders. Consequently, their role as therapeutic targets is widely explored. To date, seven different sirtuins (SIRT1-7) have been found in mammals, with SIRT1 and SIRT2 being the most studied. Nicotinamide (1), EX-527 (2) and 2-anilinobenzamide (3) have been reported as SIRT inhibitors (Figure 1). In an effort to discover alternative and selective nicotinamide pocket (C-pocket) sirtuin inhibitors, we decided to prepare and screen a small-molecule compound library based around the 10,11-dihydro-5H-dibenzo[b,f]azepine scaffold (4), structurally related to the above known SIRT inhibitors. Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme Successfully, this approach led us to the identification of novel, highly selective low-micromolar SIRT2 inhibitors. In vitro screening assays revealed our best hit (5) to have an IC50 of 18 !M against SIRT2 and high selectivity over the SIRT1 isoform (Figure 2). Cellular screening assays, performed on MCF-7 cells, confirmed the in vitro selectivity and showed compound 5 to have antiproliferative activity at a concentration of 30 !M. Finally, docking studies with compound 5 were performed to predict its binding mode at the SIRT2 catalytic site and rationalise the observed isozyme selectivity. Further biological evaluation of hit molecule 5 is currently underway. 2) Towards the Discovery of Novel HDAC4 Inhibitors The class IIa HDAC4 enzyme is under extensive investigation, since its cellular functions have potential implications in human disease. Recently, it has been found that HDAC4 levels are significantly increased in platinum-resistant ovarian cancer cells and silencing of HDAC4 in those cell lines restores platinum sensitivity. Inhibition of this HDAC isozyme with pharmacological intervention could therefore potentially reverse clinically acquired platinum resistance in cancer cells. Hence, selective HDAC4 inhibitors have the potential to represent a novel class of anticancer agents, useful in combination therapy with other HDAC inhibitors or conventional chemotherapeutic agents. To date, only a limited number of HDAC4 inhibitors have been discovered and they all show dose-limiting toxicity, poor pharmacokinetics and metabolic instability, mainly due to the presence of reactive zinc-binding groups, such as the trifluoromethylketone and the hydroxamic acid moieties (Figure 3). In order to identify novel, non-toxic and selective HDAC4 inhibitors, we considered a novel molecular framework, bearing an alternative, drug-like zinc-binding group. A small compound library was synthesised and evaluated for its potential to inhibit the HDAC4 isoform, and restore platinum sensitivity in two different types of platinum-resistant ovarian cancer cell lines: PEO4 and SKOV3. Disappointingly, functional screens showed no HDAC4 inhibitory activity for the newly designed compounds, however a few molecules were found to be active in cellular assays. Remarkably, compounds 10 and 11 (Figure 4) were able to resensitise PEO4 and SKOV3 platinum-resistant ovarian cancer cell lines at low-micromolar concentrations, without showing any general cytotoxicity, even at concentrations up to 100 μM. Additional biological investigation is planned, with the aim to gain insights into the molecular mechanisms underlying this novel class of compounds.

HDAC inhibitors are known to have anti-inflammatory properties. HDAC inhibitors are used in combination with Oct4 to generate induced pluripotent stem cells. I hypothesized that polyesters based on simple aliphatic HDAC inhibitors like valproic acid (VPA) and phenylbutyric acid (PBA) can serve as alternatives to existing polyester biomaterials with improved anti-inflammatory properties and as scaffolds for generation of iPSCs when used in combination with layer-by-layer thin films delivering reprogramming transcription factors. Vinyl ester of phenylbutyric acid (VEPA) and vinyl ester of valproic acid (VEVA) were synthesized from their carboxylic acid precursors using an iridium complex catalyst at yields as high as 97% and 73%, respectively. Amorphous poly(VEPA) and poly(VEVA) polymers were prepared by free radical solution polymerization and characterized for molecular weight and glass transition temperature. Poly(VEPA) and poly(VEVA) microparticles of 20-40 um diameter were prepared by an emulsion-solvent evaporation method and examined under scanning electron microscopy (SEM). Their hydrolytic degradation was studied by dry weight loss and HDAC inhibitor release via high performance liquid chromatography (HPLC) in the presence of varied pH and lipase-containing buffers. No significant degradation occurred within 5 days, and an MTT assay conducted on HeLa cells in the presence of these microparticles confirmed an absence of cytotoxicity. Poly(VEPA) and poly(VEVA) microparticles were not found to be a suitable biomaterial for hypothesized applications in light of their poor degradation characteristics in vitro.

The study of the nucleon structure has been a major research focus in fundamental physics in the past decades and still is the main research line of the Thomas Jefferson National Accelerator Facility (Jefferson Lab). For this purpose and to obtain statistically meaningful results, a highly efficient polarized target is essential. This means high polarization and high relative density of polarized material. This dissertation presents the principles and usage procedures of a Hydrogen Deuteride (HD) target that presents both such characteristics. Although the HD target has been shown to work successfully under a high intensity photon beam, it remained to be seen if the target could stand an electron beam of reasonably high current (nA). In this perspective, the HD target was tested for the first time in its "frozen spin" mode under an electron beam during the g14 experiment in the Jefferson Lab's Hall B in 2012. Two methods of polarimetry are also discussed in this dissertation : one with Nuclear Magnetic Resonance of this HD target during the electron beam tests, and another with the elastic scattering of electrons off a polarized target by using data taken on helium-3 during the E97-110 experiment that occurred in Jefferson Lab's Hall A in 2003.

Background: The higher-order structure of chromatin changes in response to extracellular and environmental signals. We observed nuclear morphological changes in biopsied cancer tissue after chemotherapy. Since chromatin structure dictates gene expression, and therefore function, further investigation of this phenomenon may increase our understanding of therapeutic responses. I hypothesised that nuclear morphological changes in cancer in response to DNA-damage by chemotherapy are mediated by histone deacetylases (de Ruijter, van Gennip et al.). Methods: Ovarian cancer cell lines PEO1/PEO4 (platinum sensitive/resistant) were selected as in vitro models, and primary ovarian cancer xenografts OV1002 and HOX424 as in vivo models. Expression levels of HDACs, heterochromatin protein 1 (HP1), and DNA damage response (DDR) proteins were profiled by Western blot analysis after treatment with cisplatin. Immunofluorescence imaging was undertaken using confocal microscopy, and nuclear texture and γH2AX foci were measured in Image J. Cell cycle and apoptosis were detected by flow cytometry. Thirty eight different ovarian cancer biopsies and 175 xenograft samples were assessed for HDAC and HP1 expression in response to chemotherapy by quantitative immunofluorescence. HDAC2 expression was modulated by interfering RNAs (siRNA). Results: I demonstrated nuclear morphological changes in clinical tumours, xenografts, and cell lines in response to platinum chemotherapy by robust measurement of nuclear texture. Expression of HDAC2 increased in PEO1 cells treated with cisplatin at 24h, and this was accompanied by high expression of HP1s. Expression of components of both HDACs and DDR pathways (pBRCA1, γH2AX, pATM, pATR) showed time dependent changes after cisplatin treatment. Knockdown of HDAC2 reduced the expression of HP1, induced DNA double strand breaks (DSB) measured by γH2AX, and interfered with the activation of DDR induced by cisplatin. Furthermore, HDAC2 depletion affected γH2AX foci formation, cell cycle distribution, and apoptosis triggered by cisplatin, and was additive to the inhibitory effect of cisplatin in cell lines. By inhibiting expression of HDAC2, I observed reversible alteration of chromatin patterns during cisplatin treatment to some degree. In clinical ovarian cancer specimens, expression of HDAC4, HDAC8 and HP1γ significantly increased after chemotherapy in sensitive patients, with enhanced heterogeneity in chromatin pattern. HDAC2, HDAC8, and HP1 expression were also increased after carboplatin treatment in carboplatin-sensitive xenografts. Conclusion: These results demonstrate alterations in nuclear morphology after chemotherapy, and implicate HDACs in having a role in higher order chromatin changes and in cellular DNA damage responses in ovarian cancer both in vitro and in vivo.

L’acétylation des histones apparaît comme un commutateur central de l’inter-conversion entre l’état permissif et répressif des domaines de la chromatine. L’homéostasie de l’acétylation des histones est assurée par les histones acétyltransférases (HAT) et les histones déacétylases (HDAC). Mais leurs rôles aux niveaux de la chromatine et de la régulation transcriptionnelle ou post-transcriptionnelle ne sont pas éclairci. Les objectifs de ce travail étaient d’étudier le rôle de l’acétylation des histones dans le contrôle de l’expression de gènes chez Arabidopsis thaliana. Pour cela, des mutants HDACs et HATs ont été identifié et caractérisé. Tout d’abord, AtGCN5 intervient, avec la voie des miARNs, aux niveaux transcriptionnel et post-transcriptionnel. Ceci indique que l’acétylation/déacétylation des histones est un mécanisme épigénétique impliqué dans la régulation de la production de miARN. La caractérisation des mutations HDACs révèle que la mutation de AtHDA9 induit un phénotype de floraison précoce en conditions de jours courts. Ces caractéristiques s’accompagnent d’une sur-expression de gènes activateurs dans la voie de la transition florale. La mutation de AtHDA9 et AtSRT2 affecte également la méthylation de l’ADN de la séquence péricentromérique répétée de 180 pb et du transposon AtSN1. Ces résultats révèlent les différents rôles joués par des HATs et HDACs individus dans le contrôle de l’expression du génome d’Arabidopsis
Histone acetylation appears as a central switch for interconversion between permissive and repressive state of chromatin domains. Homeostasis of histone acetylation is made sure by histones acetyltransferases (HAT) and histones desacetylases (HDAC). But their role on chromatin and transcriptional/posttranscriptional regulation was not clear. The objective of my work was to study the role of histone acetylation in the control of gene expression in Arabidopsis thaliana. For this reason, HAT and HDAC mutants had been identified and characterized. First of all, we show that AtGCN5 interfere, in the pathway of miRNA, on transcriptional and posttranscriptional regulation of gene. It indicates that histone acetylation/desacetylation is an epigenetic mechanism involved in the regulation of miRNA production. Characterization of the mutants reveled that AtHDA9 mutation induces a phenotype of early flowering in short days. This characteristics is associated with overexpression of activator genes in the pathway of flowering. AtHDA9 and AtSRT2 mutation affect also DNA méthylation of pericentromeric sequence repeat 180 bp and retroelement AtSN1. Our results reveal the different role of individual HAT and HDAC in the control of genome expression of Arabidopsis

Les aculéatines et FR235222 sont deux familles de molécules d’origine naturelle qui agissent de façons très efficaces sur les parasites intracellulaires de la famille des apicomplexes, responsables notamment du paludisme ou de la toxoplasmose. Dans la première partie de ces travaux, nous avons développé une nouvelle réaction cascade d’oxydation phénolique en « un pot » utilisant un réactif à base d’iode hypervalent en quantité catalytique. Cette stratégie de synthèse flexible et hautement modulable en peu d’étapes, permettra d’accroître l’accessibilité vers de nouveaux analogues aculéatines bioactives. Dans la deuxième partie, nous nous sommes intéressés à FR235222, un HDACi (inhibiteur des histones désacétylases) qui cible les HDACs de la classe I chez l’homme et l’HDAC3 chez le parasite T. gondii responsable de la toxoplasmose. Les HDACs sont des protéines qui jouent un rôle important dans le contrôle des mécanismes épigénétiques. Dans cette étude, un analogue fluorescent du produit naturel a été synthétisé et a permis de confirmer la cible de FR235222 chez l’homme grâce à des études de localisation cellulaire. Par la synthèse et l’évaluation de l’activité HDACi de nouveaux analogues, associées à des études de modélisation moléculaire, nous avons démontré pour la première fois que la tête chélatante céto-hydroxyle et la flexibilité du linker sont responsable de cette sélectivité sur les HDACs de la classe I humaine. Enfin, grâce à ses résultats et à l’identification de différences structurales entre l’HDAC3 humaine et parasitaire, des études de prédiction (docking) ont permis de dégager des caractéristiques essentielles d’un HDACi potentiellement sélectif sur les parasites apicomplexes
Aculeatins and FR235222 are two families of natural products that are highly effective against apicomplexan parasites, responsible for malaria and toxoplasmosis. In the first part of this work, we developed a new reaction involving a “one pot” phenolic oxidation cascade sequence using a hypervalent iodine reagent in catalytic condition. This flexible synthetic strategy will increase accessibility to new aculeatin derivatives to achieve bioactive compounds. In the second part, we were interested in FR235222, an HDACi (histone deacetylase inhibitor) which targets the class I human HDAC and parasite T. gondii HDAC3 responsible for toxoplasmosis. HDACs are proteins that play an important role in the control of epigenetic mechanisms. In this study, a FR235222 fluorescent derivative was synthesized to confirm the FR235222 target in human cells through cellular localization studies. The synthesis and assessment of the activity of new HDACi analogues, combined with molecular modelisation studies, allowed to demonstrate for the first time that the keto-hydroxyl zinc binding group and the flexibility of the linker would be responsible for this selectivity on human class I HDACs. Finally, with these results in hand and the identification of structural differences between human and parasitic HDAC3, prediction studies (docking) have revealed structural determinants to design inhibitors selective for apicomplexan parasites HDACi

Le développement embryonnaire est un processus hautement contrôlé où différentes voies de signalisation se coordonnent pour la construction d'un organisme. L'une des principales voies de signalisation impliquées dans ce processus est la voie canonique Wnt. La longue quête pour comprendre la cascade de signalisation Wnt/β-catenine a révélé que la réponse transcriptionelle induite par le signal Wnt/β-catenine est dépendante du contexte, ou compétence, cellulaire. Peu de choses sont connues sur les évènements moléculaires qui influencent cette compétence cellulaire. Dans les embryons de X. laevis Wnt/β-catenine est le signal inducteur pour l'Organisateur de Spemann. On ne sait pas ce qui limite l'activité Wnt dans ce territoire et par voie de conséquence la taille de l'Organisateur. Les résultats présentés dans ce manuscrit de thèse montrent que le facteur de transcription Barhl2 affecte le développement de l'organisateur de Spemann. Nous démontrons que Barhl2 inhibe l'activité Wnt via son interaction avec le corépresseur Groucho et le facteur de transcription Tcf, et mobilise l'activité de Hdac1 qui agit sur la structure chromatinienne. En utilisant des expériences in vitro et in vivo sur des cellules en culture et des embryons de Xénope nous démontrons que la régulation de Barhl2 sur les activités Groucho-Tcf est maintenue pendant l'embryogenèse et joue un rôle dans le confinement des progéniteurs neuraux dans le cerveau. Ensemble, nos résultats fournissent un mécanisme nouveau et important agissant sur le contrôle de l'activité transcriptionelle Wnt et la compétence des cellules à répondre à ce signal
Embryonic development is a highly controlled process where different signaling pathways participate into the elaboration of an organism. One of the main signaling pathways is the Wnt canonical pathway. The long-lasting search to understand Wnt/β-catenin transduction cascade revealed that the net transcriptional read out of Wnt/β-catenin signaling is highly dependent on the cellular context. In X. laevis embryos Wnt/β-catenin signaling is the informative signal for the Spemann Organizer induction. However little is known on what limits Wnt activity in this territory and consequently the size of the Spemann Organizer. The results presented in this manuscript provide evidence that the evolutionarily conserved transcription factor Barhl2 limits the development of the Spemann organizer. In this territory Barhl2 inhibits Wnt activity via its interaction with the co-repressor Groucho and the transcription factor Tcf. It participates to the recruitment of the chromatin remodeling enzyme, Hdac1 that represses the expression of Spemann organizer genes. Using a Xenopus tropicalis Tcf reporter line we demonstrate that Barhl2 inhibitory effect on Groucho-Tcf activities is maintained during embryogenesis and plays a role in the confinement of neural progenitors in the brain. Together, our results provide a new and important mechanism for the control of Wnt transcriptional activity

Det finns en kedja till en bra välfärd. Alla individer har behov och önskningar. Dessa ska tillgodoses med de resurser som landet får genom sin årliga produktion av varor och tjänster, BNP. Hur resurserna ska fördelas är en politisk fråga. Det är svårt att mäta välfärd. Några problem kan vara:"Reducering av verklighetens pluralism och mångfald"; Individernas olika preferenser Olika välfärdsmått är bruttonationalprodukten (BNP), Human Development Index (HDI) och Physical Quality of Life Index (PQLI). Där BNP är rent baserat på produktion medan HDI och PQLI är sammansatta index med olika indikatorer. Min analys går ut på att jämföra 3 i-länder samt 3 u-länder för att urskilja något samband mellan dels tillväxt och HDI. Jag söker också ett svar på hur storleken på de offentliga utgifter påverkar tillväxten samt HDI. Tillväxten har ökat i så väl i-länderna Sverige, USA och Japan som u-länderna Haiti, Bangladesh och Nigeria. HDI har dock minskat i i-länderna men ökat i u- länderna. Min analys visar att Japan är det enda land av de tre observerade i- länderna som har ökat sin offentliga konsumtion. Sverige och USA har minskat sin offentliga konsumtion mellan åren 1987 och 1997.

O desenvolvimento tumoral é um processo que compreende diversas etapas e consiste no desenvolvimento progressivo de células normais para um estado neoplásico através de diversas mudanças bioquímicas e fenotípicas. Entre as principais marcas do câncer estão a capacidade de invasão tecidual e metástase. A metástase é responsável por, aproximadamente, 90% das mortes causadas por câncer. Assim, os métodos mais efetivos para a melhoria dos índices de morbidade e mortalidade relacionados ao câncer são a detecção precoce, a prevenção e o tratamento da metástase. O processo de EMT, que ocorre naturalmente durante a embriogênese e reparo tecidual, está envolvido também na progressão e metástase do câncer. A EMT induz alterações celulares e microambientais complexas que resultam na aquisição de um fenótipo mesenquimal pelas células epiteliais, juntamente com um aumento das capacidades migratórias e invasivas celulares. A EMT pode ser induzida por diversos fatores extracelulares, como os fatores de crescimento TGF?, EGF, HGF e PDGF. Além disso, a superexpressão de fatores de transcrição como SNAIL, SLUG, ZEB1 e TWIST1 também é capaz de induzir a EMT in vitro. A fim de ampliar o conhecimento dos mecanismos envolvidos no processo de EMT a nível de proteínas, foram realizadas neste trabalho análises de alterações proteômicas em diferentes modelos de indução da EMT na linhagem de adenocarcinoma de mama MCF7, sendo eles a superexpressão do FT SNAIL, o tratamento com o inibidor de histonas deacetilases SAHA e o tratamento com o fator de crescimento EGF. A análise proteômica detalhada por LC-MS/MS das frações subcelulares de núcleo, citoplasma e membrana das células superexpressando SNAIL gerou uma lista de proteínas reguladas relacionadas com o processo de EMT e que foram avaliadas nos demais modelos de indução. Entre essas, a proteína HDAC1, que teve seus níveis diminuídos pela superexpressão de SNAIL. O tratamento da linhagem MCF7 com o inibidor de histonas deacetilases SAHA demonstrou uma correlação positiva com o aumento dos níveis de SNAIL nas células MCF7, sugerindo um cross-talk entre ambas as proteínas. Além disso, otratamento com SAHA induziu alterações celulares e proteicas que também sugerem a indução do processo de EMT nas células MCF7. Por fim, o tratamento com o fator de crescimento EGF também foi capaz de induzir a EMT nas células MCF7 e apresentou envolvimento na regulação do ciclo celular, alterações de proteínas em comum com os demais tratamentos e regulação diferencial de proteínas entre os subcompartimentos, indicando similaridades entre os processos e potenciais mecanismos de translocação subcelular. Em conclusão, este estudo relevou proteínas alvo relacionadas à EMT, abrindo possibilidades para tentar alterar processos relacionados à progressão tumoral e ao processo metastático.
Tumor development is a process comprising several steps and consists in the progressive development of normal cells into a neoplastic state through various biochemical and phenotypic changes. Among the major marks of cancer are the capacity for tissue invasion and metastasis. Metastasis accounts for approximately 90% of cancer deaths. Thus, the most effective methods for improving cancer-related morbidity and mortality rates are early detection, prevention and treatment of metastasis. The EMT process, which occurs naturally during embryogenesis and tissue repair, is also involved in cancer progression and metastasis. EMT induces complex cellular and microenvironmental changes that result in the acquisition of a mesenchymal phenotype by epithelial cells, together with an increase in migratory and invasive cellular capacities. EMT can be induced by various extracellular factors, such as TGF?, EGF, HGF and PDGF. In addition, overexpression of some transcription factors such as SNAIL, SLUG, ZEB1 and TWIST1 is also capable of inducing EMT in vitro. In order to increase the knowledge of the mechanisms involved in the EMT process, we performed proteomic analysis in different models of EMT induction in the MCF7 breast adenocarcinoma cell line, which were the overexpression of SNAIL, treatment with the histone deacetylase inhibitor SAHA and treatment with the growth factor EGF. The detailed proteomic analysis by LC-MS/MS of the subcellular fractions of nucleus, cytoplasm and membrane of the overexpressing SNAIL cells generated a list of regulated proteins related to the EMT process and that were evaluated in the other models of induction. Among these, the HDAC1 protein, which had its levels decreased by SNAIL overexpression. Treatment of the MCF7 cell line with the histone deacetylase inhibitor SAHA showed a positive correlation with the increase of SNAIL levels, suggesting a cross-talk between both proteins. In addition, SAHA treatment induced cellular and protein alterations that also suggest the induction of the EMT process in MCF7 cells. Finally, the treatment with the growth factor EGF was also able to induce the EMT in MCF7 cells and showed involvement in the regulation of the cell cycle, changes in proteins in common with the other treatments and differential regulation of proteins among thesubcompartiments, indicating similarities between the processes and potential mechanisms of subcellular translocation. In conclusion, this study revealed target proteins related to EMT, opening possibilities to try to alter processes related to tumor progression and metastatic process.

Statins are widely used to lower cholesterol and thus reduce cardiovascular risk, although they increase diabetes risk. Butyrate, which is produced by gut bacteria and alters gene expression through epigenetic means, lowers cholesterol and protects against diabetes in animals. This thesis compares the effects of statins and butyrate on epigenetics, cholesterol metabolism and glucose metabolism in cultured cells. Both statins and butyrate lowered cellular cholesterol and impaired insulin secretion, although they acted through different mechanisms.

A esquistossomose é um grave problema de saúde pública, com alta mortalidade e morbidade em países endêmicos, causada pelo verme trematódeo do gênero Schistosoma. O praziquantel é a única droga disponível para tratamento da doença, é usada em larga escala para tratamento de populações de áreas endêmicas, porém não previne a reinfecção e tem efeito somente em vermes adultos. Drogas estudadas em câncer como inibidores de histona deacetilases (iHDACs) modificam o padrão epigenético da célula desencadeando a morte celular, e em Schistosoma mansoni já foi mostrado que a inibição de HDACs além de aumentar a acetilação de histonas alterou o fenótipo de miracídios e provocou morte em esquistossômulos e vermes adultos. O presente estudo investigou o efeito do iHDAC Trichostatin A (TSA) na regulação da transcrição gênica em esquistossômulos, detectando por meio de ensaios de microarray centenas de genes diferencialmente expressos, relacionados a replicação de DNA, metabolismo e complexos modificadores de histonas. A inibição de HDAC em vermes adultos levou a um aumento da acetilação nas marcas de histonas H3K9ac, H3K14ac e H4K5ac relacionadas à indução de transcrição. Com imunoprecipitação de cromatina seguida de PCR (ChIP-qPCR) detectou-se o aumento de deposição de H3K9ac e H3K14ac na região promotora de genes com expressão aumentada ou diminuída, porém a marca de repressão H3K27me3 não sofreu alteração na região promotora de nenhum gene analisado. Análises adicionais indicaram um conjunto de genes diferencialmente expressos que codificam proteínas histone readers, que fazem parte de complexos modificadores de histonas, como EED capaz de identificar a marca de repressão H3K27me3 e regular a atividade de EZH2, apontando um novo alvo terapêutico. O efeito sinérgico entre iHDAC e um iEZH2 foi testado e detectou-se o aumento da mortalidade de esquistossômulos. A estrutura de SmEZH2 foi modelada por homologia e usada para análises computacionais que sugeriram uma alta afinidade de ligação de SmEZH2 com o iEZH2, abrindo uma perspectiva de desenvolvimento de novas drogas específicas para tratamento da esquistossomose.
Schistosomiasis is a serious public health problem, with high mortality and morbidity in endemic countries, caused by trematode worms of the genus Schistosoma. Praziquantel is the only available drug for treatment of the disease; it is used extensively to treat populations in endemic areas, but does not prevent reinfection and is effective only in adult worms. Drugs studied in cancer as histone deacetylase inhibitors (iHDACs) modify the epigenetic status of the cell, triggering cell death, and it has been shown in Schistosoma mansoni that inhibition of HDACs increase histone acetylation, alter the phenotype of miracidia and cause death in schistosomules and adult worms. The present study investigated the effect of iHDAC Trichostatin A (TSA) on the regulation of gene transcription in schistosomules, detecting by means of microarray assays hundreds of differentially expressed genes related to DNA replication, metabolism and histone remodeling complexes. Inhibition of HDAC in adult worms led to an increase in histone acetylation marks H3K9ac, and H3K14ac H4K5ac related to transcriptional induction. With chromatin immunoprecipitation followed PCR (ChIP-qPCR) we detected an increased deposition of H3K9ac and H3K14ac at the promoter region of genes with increased or decreased expression, but the repressive mark H3K27me3 was not changed at all analyzed gene promoter regions. Additional analysis indicated a set of differentially expressed genes that encode histone reader proteins that are part of histone modifier complexes such as EED, which is able to identify the repression mark H3K27me3 and to regulate EZH2 activity, pointing to a new therapeutic target. The synergistic effect between iHDAC and one iEZH2 has been tested and found to cause an increase in schistosomules mortality. The SmEZH2 structure was modeled by homology and used for computational analyses, which suggested a high affinity binding of SmEZH2 with iEZH2, opening the opportunity for development of new specific drugs for treatment of schistosomiasis.

Doctor of Philosophy
Psychological Sciences
Mary E. Cain
Gene-environment interactions play a significant role in drug abuse and addiction. Epigenetics (the study of how environmental stimuli alter gene expression) has gained attention in recent years as a significant contributor to many behavioral phenotypes of drug addiction. The current study sought to determine if differential rearing conditions can alter a specific epigenetic mechanism, histone deacetylase (HDAC), and how HDAC inhibition can affect drug-taking and drug-seeking behaviors differently among enriched, isolated, or standard-housed rats. Ninety male Sprague-Dawley rats were reared for 30 days in enriched (EC), isolated (IC), or standard (SC) conditions prior to amphetamine (0.03, 0.05, 0.1 mg/kg/infusion, i.v.) self-administration, extinction, or reinstatement sessions. Trichostatin A (TsA; 0.3 mg/kg, i.v.), an HDAC inhibitor, was injected 30 min prior to drug-taking or drug-seeking sessions. Results indicated that EC rats self-administered less amphetamine (0.03 mg/kg/infusion) than IC rats. No significant effects of TsA administration were found on general self-administration for any of the three amphetamine doses. While enrichment facilitated the extinction of active lever pressing, there was also a mild facilitation of extinction in IC-TsA rats compared to IC-vehicle counterparts. Lastly, TsA administration decreased cue-, but not drug-induced reinstatement, with IC-TsA rats exhibiting significantly attenuated cue-induced reinstatement compared to IC-vehicle rats. These findings suggest that differential rearing can alter HDAC mechanisms that can change drug-seeking behaviors, particularly in rats reared in isolated conditions. While TsA-induced HDAC inhibition may be less protective against general amphetamine self-administration, it may decrease drug-seeking tendencies during relapse that are induced by the reintroduction of contextual environmental cues heavily associated with drug reward.

Therapeutic responses to Histone deacetylase (HDAC) inhibitors (HDACi) in many cancers are well described but development of resistance to HDACi is a major stumbling block. Whether HDACis induce epigenetic reprogramming and how this contributes to relapse is not reported. A CTCL cell line HuT78, and a CLL cell line MEC1, were used to develop HDACi resistant clones (RHuT78 and RMEC1 respectively) that persistently grow in the presence of the clinically used HDAC inhibitor Romidepsin. RHuT78 cells show perturbed trimethylation of histone H3 lysine K4 on Romidepsin treatment which linked to higher protein expression levels of the implicated demethylase KDM5A. Following on from these experiments, a qRT-PCR epigenetic gene expression array was used to quantify levels of 84 epigenetic gene transcripts in RHuT78 cells and significantly altered genes were taken forward for further investigation. Studies of gene expression patterns in parental, resistant and ‘drug holiday’ cell lines of both HuT78 and MEC1 led to particular interest in HDAC8, DNMT3A and DNMT3B. Functional studies showed that HDAC8 overexpression increased proliferation and resistance of HuT78 cells to Romidepsin. Parallel observations suggested an increase in proliferation of resistant cell lines cultured in the presence of the HDACi. This increased proliferation was seen even with lower concentrations of Romidepsin and argues against prolonged monotherapy using HDACis. Significantly, inhibitors of DNA methyltransferases synergised with Romidepsin in a dose and schedule dependent manner, reversing the changes in epigenetic gene expression associated with resistance and causing increased apoptosis in RHuT78 cells. Taken together this thesis identifies and characterises an unacknowledged contribution of epigenetic reprogramming to drug resistance and provides insights into the effects of Romidepsin on the epigenome that could potentially contribute to HDACi resistance.

3,4-methylenedioxymethamphetamine (MDMA) is a psychoactive substance used for recreational purposes. Possible clinical use of MDMA, in combination with psychotherapy, has been considered for the treatment of PTSD. However, MDMA causes neurotoxic effects and its use was associated with psychiatric symptoms, memory and cognitive deficits. To elucidate these aspects, the first aim of this study was to investigate the effects of acute and repeated MDMA treatment on BDNF/TrkB and HDACs in animal models. According to recent evidence about HDAC inhibitors, we used sodium butyrate to investigate its ability to affect MDMA-induced molecular and behavioral alterations. Moreover, considering that an alteration of BDNF has been reported in the brain of animals treated with psychoactive substances and in the blood of substance abusers, possible alterations of this neurotrophin levels were investigated in blood samples of MDMA users. Since different BDNF pools exist in plasma and serum, distinct determination of the neurotrophin were evaluated in both matrices. Furthermore, recent evidence has shown that neurofilaments can represent valid biomarkers of neural damage. Given the neurotoxic effects of ecstasy, we investigated neurofilaments in serotonergic neuronal cells. Particularly, we assessed MDMA effects on neurofilament proteins in differentiated serotonergic cells and we investigated if BDNF could protect serotonergic neurons from MDMA effects. Data showed that MDMA alters different crucial genes as well as the proteins involved in both substance use disorders and psychiatric conditions. Animal studies showed alterations both in BDNF pathways and in HDACs. Moreover, investigation in humans brought into view that peripheral BDNF could not reflect central BDNF and serum and plasma BDNF can express different types of this neurotrophin. Furthermore, data obtained in the differentiated serotonergic cell line highlight the useful role of NF-L as a biomarker of neuronal damage induced by MDMA, confirming the importance of studying NFs in the field of neuropsychiatric disorders.

O glioblastoma (GBM) é considerado um dos tumores mais agressivos do sistema nervoso central (SNC). Mesmo com o uso de protocolos modernos de tratamento o prognóstico se mantém bastante reservado, sendo que crianças com GBM apresentam uma sobrevida média de 12 a 15 meses. Mecanismos epigenéticos podem interferir no processo de carcinogênese, sendo que a acetilação do DNA pode modular a expressão de genes que atuam no controle do ciclo celular, contribuindo assim para o desenvolvimento e progressão de neoplasias. Estudos clínicos demonstram que inibidores de histonas deacetilases (HDACs), em monoterapia ou combinados a outros agentes antineoplásicos, são clinicamente ativos e bem tolerados no tratamento de uma ampla variedade de tumores. Estes inibidores podem sensibilizar a resposta celular à irradiação ionizante, possibilitando uma redução nas doses-padrão utilizadas, minimizando os efeitos colaterais a curto e longo prazo. A radiação ionizante induz dano no DNA e é geralmente aceito que quebras da dupla-fita (DSBs) é o tipo de lesão mais severa relacionada à sobrevivência celular e preservação da integridade genômica. No presente estudo, avaliamos o potencial efeito radiosensibilizante do PCI-24781, um novo e potente pan-inibidor de HDAC nas linhagens celulares de GBM pediátrico SF188 e KNS42. Foram comparadas as taxas de proliferação celular, clonogenicidade e apoptose das linhagens SF188 e KNS42 com ou sem tratamento com PCI-24781. Também foram comparadas as taxas de clonogenicidade das linhagens SF188 e KNS42 que foram irradiadas com ou sem tratamento prévio com PCI-24781. Adicionalmente, foram avaliados os efeitos do PCI-24781 na expressão de algumas das principais proteínas responsáveis pelo reparo de quebras da dupla-fita ocasionadas pela irradiação. Para os ensaios de proliferação celular foram utilizados os tempo de 24, 48, 72 e 96h, para apoptose, 48h e para capacidade clonogênica sem irradiação o tempo de 48h, em diferentes doses de PCI-24781 (0,25 - 16 M). O inibidor bloqueou significativamente a proliferação celular (p<0,05), induziu morte por apoptose (p<0,05) e reduziu a capacidade na formação de colônias (p<0,001) em ambas as linhagens. No ensaio para avaliação da radiosensibilidade, foram utilizadas as doses do IC30 11 de cada linhagem do ensaio clonogênico seguida de diferentes doses de irradiação. Ambas as linhagens apresentaram uma significativa (p<0,001) diminuição na formação de colônias em todas as doses de irradiação. A linhagem mais resistente à droga, SF188 foi escolhida para estudo do reparo de quebras da dupla-fita ocasionadas pela irradiação. As expressões da proteína Rad51, importante na via de reparo por recombinação homóloga (HR), e das proteínas DNA-PKcs, Ku70 e Ku86, importantes na via de reparo por união terminal não-homóloga (NHEJ) apresentaram uma maior diminuição quando a linhagem irradiada foi previamente tratada com PCI-24781 em comparação à radioterapia exclusiva. Estes achados demonstram que o inibidor de histona PCI-24781 apresenta um importante papel como agente radiosensibilizante, comprometendo o reparo das quebras de dupla-fita em células de GBM pediátrico tratadas com radioterapia.
Glioblastoma (GBM) is considered one of the most aggressive tumors to affect the central nervous system (CNS). Even employing modern treatment protocols the prognosis remains very poor, with children affected by GBM presenting a median survival rate of 12 to 15 months. Epigenetic mechanisms may interfere with the process of tumorigenesis, and DNA acetylation can modulate the expression of genes that contribute in cell cycle control and participate to the development and progression of cancer. Clinical studies demonstrate that histone deacetylase inhibitors (HDACs), alone or in combination with other antineoplastic agents, are clinically active and well tolerated in the treatment of a wide variety of tumors. These inhibitors may sensitize the cellular response to ionizing radiation, enabling the reduction in standard doses of radiation, ultimately minimizing both short and long-term side effects. Ionizing radiation induces DNA damage and it is generally accepted that the double-stranded breaks (DSBs) is the most severe type of injury related to cell survival and preservation of genomic integrity. In the present study, we evaluated the potential radiosensitizer effect of PCI-24781, a novel potent pan-HDAC inhibitor in the pediatric GBM cell lines SF188 and KNS42. We compared the cell proliferation rates, apoptosis of clonogenicity of KNS42 and SF188, with or without treatment with PCI-24781. Moreover, clonogenicity rates were compared between cell lines that were irradiated with or without prior treatment with PCI-24781 Additionally, we evaluated the effects of PCI-24781 in the expression of some of the major proteins responsible for the repair of double-stranded breaks caused by the irradiation. For the cell proliferation assays, the times of 24, 48, 72 and 96 hours were used, for apoptosis, the time of 48h and clonogenic capacity without irradiation, the time of 48h, and different doses of PCI-24781 (0,25 - 16 M). The inhibitor significantly blocked cell proliferation (p<0,05), inducing cell death by apoptosis (p<0,05) and reducing the colony forming ability (p<0,001) of both lineages. In the assays to evaluate the radiosensitivity , the IC30 doses of the clonogenic assays were used for each cell-line after different doses of irradiation. Both lineages showed a significant decrease (p<0,001) in colony formation at all doses of irradiation. The most resistant cell-line to the drug, SF188, was 13 chosen to study the double-strand breaks repair caused by irradiation. The Rad51 protein levels, critical for homologous recombination (HR), and the DNA-PKcs proteins Ku70 and Ku86, important for DNA repair through non-homologous end joining (NHEJ) showed significant decrease in expression when cell-line was treated with PCI-24781 prior to radiotherapy. These data demonstrates that the histone deacetylase inhibitor PCI-24781 plays an important role as a radiosensitizer agent, compromising the repair of double-strand breaks in pediatric GBM cells following irradiation.

Epigenetic therapies of cancer have been intensely studied, because of the high frequency of epigenetic alterations found in tumor cells. Epigenetic modulators, such as histone deacetylases (HDAC) and DNA methyltransferase (DNMT), can be targeted by specific drugs with the intent to revert the epigenetic alterations induced by tumor progression. Although a few DNMT inhibitors have been approved for the therapy of some specific hematopoietic diseases, most of the results of clinical trials on other types of cancer have given limited results. The need for a better understanding of the molecular mechanism(s) underlying sensitivity to epigenetic therapies is therefore very strong. For obvious reasons, the use of preclinical models is mandatory to achieve this deeper understanding, to be translated to better clinical trials. We therefore used one form of acute myeloid leukemias (APL) as a preclinical model for epigenetic therapies. The choice of APL is due to the fact that the APL-associated fusion protein PML/RARα is known to induce epigenetic alterations in APL blasts by recruiting HDACs and DNMTs, making APL blasts obvious candidates for this kind of drugs. Monotherapies of either HDACi (VPA) or DNMTi (DAC) induced increased survival in APL mice and their combination were able to further prolong survival of APL mice, demonstrating the importance of hitting multiple targets of the epigenetic “Silencing Loop”. Both the studied epigenetic drugs also demonstrated an enhancement of the therapeutic effect of differentiating therapy with All Trans Retinoic Acid (ATRA). We also tried to identify the molecular mechanisms involved in anti-leukemic activity. Both VPA and DAC demonstrated an induction of the apoptotic response in leukemic cells through the upregulation of members of the Death Receptor pathways. When treating with VPA or DAC, we also observed some modifications in chromatin structure and DNA methylation pattern in a specific CpG rich region within the TRAIL promoter. These results suggest a partial direct epigenetic mechanism underlying TRAIL induction by DAC and VPA. We were able to confirm the results obtained in the APL model in an unrelated form of AML derived from the expression of AML1/ETO fusion protein, suggesting that a substantial fraction of AML patients could benefit from epigenetic treatments, and further reinforcing our view that the APL model represents a paradigm for the study of epigenetic drugs.

NTECEDENTS: els inhibidors d’histona deacetilasa (HDACi) han emergit els últims anys com potencials tractaments dirigits, amb la finalitat de revertir els canvis epigenètics associats a diferents tipus de càncer, incloent les neoplàsies hematològiques. La falta d’especificitat dels HDACi, però, dificulta la predicció dels seus efectes biològics i provoca una toxicitat no menyspreable, el que ha limitat el seu ús en la pràctica clínica. L’expressió de les HDACs no ha estat estudiada en les leucèmies agudes pediàtriques més que de manera parcial i en sèries amb un limitat número de pacients; per tant, són necessaris estudis que ajudin a aclarir el paper de cadascuna de les HDACs com a possibles biomarcadors i com a dianes terapèutiques, de cara a dirigir el tractament específic i personalitzat amb HDACi. HIPÒTESI: en aquest context, l’estudi global del perfil transcripcional de les HDACs en una sèrie àmplia i representativa de pacients pediàtrics amb diferents subtipus de leucèmia aguda permetria definir millor el seu impacte pronòstic i definir el seu rol com a dianes terapèutiques, ajudant a dirigir el tractament específic amb HDACi de forma més racional i individualitzada. OBJECTIUS: estudiar de forma global el perfil d’expressió de les HDAC1-11, SIRT1, SIRT7 i d’altres gens co-reguladors, MEF2C i MEF2D, en una sèrie de pacients pediàtrics amb leucèmia aguda diagnosticats i tractats en un sol centre. METODOLOGIA: anàlisi de l’expressió de l’mRNA de les HDAC1-11, SIRT1, SIRT7, MEF2C i MEF2D mitjançant PCR quantitativa en 211 pacients pediàtrics (0-18 anys), diagnosticats de leucèmia de novo des de l’any 2003 al 2017 en el nostre centre. Els pacients es van tractar uniformement d’acord als protocols consecutius de la Sociedad Española de Hematología y Oncología Pediátrica (SEHOP). Es va emprar un pool de pacients no-neoplàsics com a calibrador i també es va analitzar l’expressió dels diferents gens en cèl·lules CD34+ normals de moll d’os i a cèl·lules B CD19+ madures i limfòcits T madurs de sang perifèrica. RESULTATS: l’expressió de les HDACs va diferir en els diferents subtipus de cèl·lules hematopoètiques normals d’acord amb el grau de maduració i el llinatge. En els pacients amb leucèmia es va observar, en general, una sobreexpressió de les HDACs, amb perfils específics que correlacionaven amb característiques clíniques i biològiques i, fins i tot en algunes ocasions, amb la supervivència dels pacients. Així, alguns perfils d’expressió d’HDAC i el perfil de MEF2C semblaven reflectir, probablement, el llinatge i la maduresa dels blasts, mentre que d’altres identificaven la via oncogènica activa en les cèl·lules leucèmiques. Concretament, es va identificar un perfil d’expressió distintiu pels pacients amb reordenament del gen KMT2A (MLL), amb nivells elevats d’HDAC9 i MEF2D, independentment de l’edat, el partner del gen de fusió i el llinatge. A més, es va observar en els pacients amb LLA-B un pronòstic advers de la sobreexpressió de l’HDAC9, independentment de l’estat del gen KMT2A. CONCLUSIONS: en aquest treball s’han observat diferents perfils d’expressió segons els diferents subtipus de leucèmia aguda pediàtrica. Concretament, s’ha identificat un perfil distintiu dels pacients amb reordenament del gen KMT2A, amb la sobreexpressió de l’ HDAC9 i MEF2D, independentment del llinatge, de l’edat i del partner del gen de fusió. Aquests resultats aporten una imatge global de l’expressió de les HDACs en els pacients pediàtrics amb leucèmia aguda i recolzen el tractament dirigit amb HDACi més específics en seleccionats grups de pacients d’alt risc, com els pacients amb reordenament del gen KMT2A.
Histone deacetylase inhibitors (HDACi) emerged as promising drugs in leukaemia, but their toxicity due to lack of specificity limited their use. Therefore, there is a need to elucidate the role of HDACs in specific settings. The study of HDAC expression in childhood leukaemia could help to choose more specific HDACi for selected candidates in a personalized approach. We analysed HDAC1-11, SIRT1, SIRT7, MEF2C and MEF2D mRNA expression in 211 paediatric patients diagnosed with acute leukaemia. We found a global overexpression of HDACs, while specific HDACs correlated with clinical and biological features, and some even predicted outcome. Thus, some HDAC and MEF2C profiles probably reflected the lineage and the maturation of the blasts and some profiles pointed out specific oncogenic pathways active in the leukaemic cells. Specifically, we identified a distinctive signature for patients with MLL rearrangement, with high HDAC9 and MEF2D expression, regardless of age, MLL-partner and lineage. Moreover, we observed an adverse prognostic value of overexpression of HDAC9, regardless of MLL rearrangement. Our results provide useful knowledge on the complex picture of HDACs expression in childhood leukaemia and support the directed use of specific HDACi to selected paediatric patients with acute leukaemia.

Abnormal epigenetic control is a common early event in tumour progression, and aberrant acetylation in particular has been implicated in tumourigenesis. Histone deacetylases (HDACs) are enzymes that regulate acetylation of chromatin and a variety of other non-histone substrates. Significantly, HDAC inhibitors are potent anti-proliferative agents and exhibit clinical activity in lymphoproliferative and haematological maligancy. However, the mechanistic details by which HDAC inhibitors affect proliferation remain to be elucidated. I have explored the cellular processes affected by HDAC inhibitors, and begun to illuminate a new pathway, regulated by HDAC, which impinges on the cellular effect of HDAC inhibitors. My results suggest that the proteins HR23B and Myd88 are important sensitivity determinants for HDAC inhibitor treatment, and that their interplay with HDAC6 dictates cell fate choice between survival by autophagy or apoptosis.