Histone phosphorylation is often a direct outcome of activated intracellular signaling pathways, and functions to translate extracellular signals into appropriate biological outputs such as changes in gene expression. Growth factors and cellular stress trigger rapid and transient expression of immediate-early genes (such as c-fos, c-jun) in mammalian cells, and their induction strongly correlates with a transient phosphorylation of S10 and S28 on histone H3. While many signaling cascades that lead to H3 phosphorylation have been mapped out, mechanistic details of the downstream events and how H3 phosphorylation contributes to transcriptional activation are still poorly defined. To investigate the direct effects of H3 phosphorylation on transcription, we targeted the H3 kinase MSK1 to endogenous c-fos promoter, and found that this is sufficient to activate its expression. Moreover, targeting MSK1 to the tissue-specific -globin gene induces H3S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Mechanistically, H3S28 phosphorylation not only disrupts binding of polycomb repressive complexes, but also induces a methyl-acetylation switch of the adjacent K27 residue. This provides the first indication that H3 phosphorylation is involved in antagonizing polycomb silencing. To further identify post-translational modifications (PTMs) that function together with MSK1-mediated H3 phosphorylation, I developed a novel nucleosome purification approach called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique combines in vivo biotinylation by BirA and H3 phosphorylation by MSK1, allowing enrichment of phosphorylated nucleosomes using streptavidin. I found that MSK1-phosphorylated nucleosomes are hyper-acetylated on H3 and H4, and importantly, I identified a trans-tail crosstalk between H3 phosphorylation and H4 acetylation on K12. This proof-of-principle study demonstrates that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes. Taken together, work described in this thesis shows that histone H3 phosphorylation can initiate additional PTM changes on other residues within the nucleosome, and such crosstalk plays an important role in regulating gene expression.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Acetylation was previously thought to occur exclusively on histones, but recent high-throughput screens have identified thousands of non-histone substrates. Despite the identification of these sites, little is known about how these acetyltransferase enzymes target their substrates. Gcn5 is the catalytic acetyltransferase found within the highly conserved SAGA complex. Recently, a member of this complex, Ada2, was found to impact Gcn5 substrate selection. In the yeast model organism Saccharomyces cerevisiae, a synthetic substrate developed from a proposed Gcn5-specific consensus sequence is used to identify regulators of Gcn5 substrate selection. This work is the first to demonstrate that addition of a consensus sequence is enough to confer acetylation of a non-substrate. With this method, Ada3 was identified as a key regulator, and acetylome profiling identified novel targets for Gcn5 dependent acetylation specifically regulated by Ada3. This system could be adapted for other acetyltransferases to identify regulators of substrate selection.

O (-)-?-bisabolol é um produto natural abundante na natureza, possui atividades biológicas reconhecidas, que o torna extremamente interessante para as indústrias de cosméticos e fármacos. Este trabalho tem como objetivo à preparação de novos compostos com possíveis aplicações biológicas, agregando valor ao produto natural. Durante as transformações procura-se trabalhar com metodologias ecologicamente corretas (Green Chemistry). O trabalho foi dividido em 4 (quatro) partes principais: oxidação, cicloadição, alquilação e acetilação. A oxidação alílica do (-)-?-bisabolol usando trióxido de cromo (CrO3) forneceu o Deodarone (tetraidro-2,2,6-trimetil-6-(4-metil-3-cicloexen-1-il)-4H-piran-4-ona) com 45,3% de rendimento. A cicloadição [1+2] entre diclorocarbeno e o (-)-?-bisabolol usando CETABr ou KF/Al2O3 originou o 4-(2,2-dicloro-3,3-dimetilciclopropil)-2- (7,7-dicloro-6-metilbiciclo[4.1.0]hept-3-il)-2-butanol com rendimentos de 92% e 97% respectivamente. A alquilação do álcool benzílico com o produto resultante da cicloadição gerou o 4-((2,2-bis(benzilóxi)-3,3-dimetilciclopropil)-2-(7,7- bis(benzilóxi))-6-metillbiciclo[4.1.0]heptan-3-il)butan-2-ol, o rendimento obtido foi de 92%. A epoxidação do (-)-?-bisabolol forneceu o Óxido de Bisabolol B: 1-metil- 1-[5-(4-metil-3-cicloexenil)tetraidro-2-furanil]etil álcool com rendimento de 30%. A acetilação do (-)-?-bisabolol utilizando DMAP ou ZnCl2 forneceu o 2-acetil-(S)-2- metil-2-((R)-4-metillciclohex-3-enil)hept-5-eno com rendimentos de 72,6% e 76,5% respectivamente. Os resultados obtidos foram bastante promissores e podem ser expandidos para trabalhar com outros grupos de moléculas com atividades biológicas. Todos os compostos foram caracterizados por RMN13C (apt). / The (-)-?-bisabolol is a natural product abundant in nature, it has recognized biological activities, which makes it very interesting for the cosmetic and pharmaceutical products. This work aims to prepare new compounds with potential biological applications, adding value to natural product. During the transformation we seek to work with eco-friendly methodologies (Green Chemistry). The work was divided into four (4) main parts: oxidation, cycloaddition, alkylation and acetylation. The allylic oxidation of (-)-?-bisabolol using chromium trioxide (CrO3) provided Deodarone (tetrahydro-2,2,6-trimethyl-6-(4-methyl-3- cicloexen-1-yl)-4H-pyran-4-one) with 45,3% yield. The cycloaddition [1+2] between dichlorocarbene and (-)-?-bisabolol using CETABr or KF/Al2O3 afforded the 4 - (2,2-dichloro-3 ,3-dimethylcyclopropyl) -2 - (7,7-dichloro-6-methylbicyclo [4.1.0] hept-3-yl)-2-butanol with yields of 92% and 97% respectively. Alkylation of benzyl alcohol with the product resulting from cycloaddition generated the 4-(2,2-bis (benzyloxi)-3,3-dimethylcyclopropil)-2-(7,7-bis(benzyloxi)) - 6 - methylbicycle[4.1.0] heptan-3-il)butan-2-ol, the yield was 92%. The epoxidation of (-)-?-bisabolol provided the Bisabolol Oxide B: 1-methyl-1-[5- (4-methyl-3-cicloexenil) tetrahydro- 2-furanyl] ethyl alcohol with yield of 30%. Acetylation of (-)-?-bisabolol using DMAP or ZnCl2 gave the 2-acetyl-(S)-2-methyl-2-((R)-4-metillciclohex-3-enyl) hept- 5-en with yields of 72,6% and 76,5% respectively. The results were very promising and can be expanded to work with others groups of molecules with biological activities. All compounds were characterized by RMN 13C (apt).

Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.

Lactate has been long thought of as a dead-end waste product of glycolysis. In the brain, recent evidence has revealed a key role of L-lactate creating a paradigm-shift in our understanding of the neuronal energy metabolism. The Astrocyte neuron lactate shuttle (ANLS) model, has shown L-Lactate as the main energy substrate delivered by astrocytes to neurons to sustain neuronal oxidative metabolism. This metabolic coupling is an essential mechanism for long-term memory formation. Experimental evidence indicates that the role of lactate in cognitive function is not limited to being a neuronal metabolic substrate, but rather it is also an important signaling molecule for synaptic plasticity. One of the new emerging roles of lactate is its effect on gene expression levels; however, our current understanding of the mechanism of lactate effect on gene expression is rudimentary. Here, I investigate the role of lactate as an epigenetic modulator in neuronal cultures. First, I explored the effect of lactate on the transcriptome and methylome of the neuronal cells using primary neuronal cell culture models. Our results reveal a significant role for lactate in inducing neuronal cell differentiation. Following, I characterized a neuroblastoma cell line as our neuronal differentiation cell model and assessed its metabolic features relative to other immortal cell lines. Further, using the cell line in vitro model, I looked into the metabolic reprograming that occurs in parallel with the first indications of differentiation, focusing on lactate production rates. Subsequently, I investigated the role of lactate in differentiation through transcriptomic analysis. We show that lactate induced histone acetylation and promoted expression of dopaminergic markers, with a stronger effect of D-lactate over L-lactate. Further studies to establish potential linkages between those two pathways will enhance our understanding of the effect of lactate.

Orientador: Andreia Bufalino / Resumo: As desordens potencialmente malignas orais (DPMOs) são apresentações clínicas que apresentam risco aumentado para o desenvolvimento de câncer na cavidade oral, seja ela uma lesão precursora definida ou mucosa clinicamente normal. Dentre as principais DPMOs, a leucoplasia oral (LO) se apresenta como uma placa branca não raspável de risco questionável, podendo ser homogênea ou não homogênea, cuja taxa de transformação maligna varia de 0,2% até 17,5%. Além disso, a LO apresenta os mesmos fatores de riscos que são observados no desenvolvimento de carcinoma espinocelular (CEC) oral, o qual é a principal neoplasia maligna que afeta a cavidade oral. Outra DPMO importante é a leucoplasia verrucosa proliferativa (LVP), a qual também se apresenta como uma placa branca não raspável, porém multifocal e possui um comportamento persistente e progressivo para malignidade, com taxa de transformação maligna de 70% até 100% dos casos. Diferente da LO, os fatores de risco como tabaco, álcool e noz de areca não parecem estar associados com o desenvolvimento da LVP. Adicionalmente, a LVP apresenta resposta inadequada a todas as modalidades de tratamento, sofre rápida disseminação pelos sítios orais e muitas vezes demonstra recorrência. Os graus de displasias identificados nas DPMOs são utilizados atualmente como preditores de risco para transformação maligna, no entanto diversos estudos têm mostrado que este não é um preditor fiel. Estudos recentes sugerem ainda que o infiltrado inflamatório asso... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Oral potentially malignant disorders (OPMDs) are clinical presentations that present an increased risk of cancer development in the oral cavity, whether in a clinically definable precursor lesion or clinically normal oral mucosa. Among the main OPMDs, oral leukoplakia (OL) presents itself as a non-scratchable white plaque of questionable risk, which can be homogeneous or non-homogeneous, whose malignant transformation rate varies from 0.2% to 17.5%. In addition, OL has the same risk factors that are observed in the development of oral squamous cell carcinoma (OSCC), which is the main malignant neoplasm affecting the oral cavity. Another important OPMD is proliferative verrucous leukoplakia (PVL), which also presents itself as a white, non-scratchable, but multifocal plaque and has a persistent and progressive behavior for malignancy, with a malignant transformation rate of 70% to 100% of the cases. Unlike OL, risk factors such as tobacco, alcohol and areca nut do not appear to be associated with the development of PVL. Additionally, PVL has a poor response to all treatment modalities, suffers rapid dissemination through oral sites and often shows recurrence. The degree of epithelial dysplasia identified in OPMDs is currently used as a risk predictor for malignant transformation, however several studies have shown that it is not a reliable predictor. Recent studies also suggest that the inflammatory infiltrate associated with OPMD may be due to its etiology and clinical compor... (Complete abstract click electronic access below) / Mestre

Capsid of mouse polyomavirus (MPyV) is composed from three structural proteins: major structural protein VP1 and minor structural proteins VP2 and VP3. Posttranslational modifications may affect functions of proteins. This work deals with acetylation of MPyV structural proteins and its impact on the viral replication cycle. First part of the thesis is focused on acetylation of VP1. We showed that the VP1 protein is acetylated in viral particles and that interaction of VP1 with minor proteins supports VP1 acetylation. Further, we showed that cytoplasmatic deacetylase, histone deacetylase 6 (HDAC6), is important for virus infectivity. Overexpression of HDAC6 decreased MPyV infectivity, also decreased infectivity was exhibited by virus isolated from HDAC6 knock out cells. In addition, VP1 protein of virus from HDAC6 knock out cells was more acetylated in comparison with virus from parental cell line. These data suggest that VP1 is substrate for HDAC6. Second part of the thesis is focused on the characterization of N-terminal acetylation of VP3 minor structural protein. It has been previously shown that VP3 protein is N-terminally acetylated and MyPV with mutated (unacetylated) form of VP3 protein is non-infectious. The main aim of this part is to prove the hypothesis that N-terminal acetylation is...

Steam exploded fibers from Yellow Poplar (Liriodendron tulipifera) wood were assessed in terms of (a) their impact on torque during melt processing of a thermoplastic cellulose ester (plasticized CAB); (b) their fiber incorporation and dispersion characteristics in a CAB-based composite by SEM and image analysis, respectively; and (c) their impact on the mechanical properties (under tension) of CAB-based composites having fiber contents of between 10 and 40% by weight. The fibers included water-washed steam exploded fibers (WEF), alkali-extracted fibers (AEF), acetylated fibers (AAEF), all from Yellow poplar (log Ro = 4.23), and oat fillers (COF) as control. The stepwise increase in cellulose content by extraction, and especially the (surface) modification by acetylation, contributed to increased torque during melt processing, and to improved interfacial adhesion as well as fiber dispersion. As compared to pure CAB, AAEF generated the highest increase in torque (+ 421%) followed by AEF (+ 260%) and WEF (+ 190%) at 40% fiber content by weight. AAEF was also found to enhance the tensile properties of the resulting composites. SEM studies of the tensile fracture surfaces indicated significant interfacial delamination and also pull - out of fibers when WEF, AEF, and COF were used to reinforce the CAB matrix. Composites with AAEF, by contrast, revealed fracture surfaces with reduced interfacial delamination and with significant fiber fracturing during failure. Image analysis was used to determine fiber dispersion within the resulting composites quantitatively. Significant improvement in fiber dispersion was achieved when the matrix was reinforced with acetylated fibers (AAEF). Fiber addition to the matrix resulted in loss of strain at break (- 80 to - 93%) and slight or significant increases in modulus (+ 47 to + 103%) depending on fiber type at 40% fiber content. Maximum stress declined for all fibers except AAEF at all fiber contents. AAEF-based composites revealed a decline in maximum stress when fiber content rose to 10%, and this reversed when fiber content increased beyond 10%. This increase in strength is consistent with the rule of mixtures that stipulates reinforcement of the matrix by fibers that are capable of transferring stresses across the fiber-matrix interface. All fibers suffered length decreases during melt processing. / Master of Science