Heterochromatin assembly in fission yeast (Schizosaccharomyces pombe) requires conserved components that mediate RNA interference (RNAi) directed methylation of histone H3 on lysine 9 (H3K9). Fission yeast heterochromatin is mainly found at centromeres, telomeres, and the mating-type locus. At centromeres, transcripts from repetitive elements are processed to siRNAs and RNAi promotes chromatin modification by recruiting the Clr4 methyltransferase. RNAi is not required to maintain silent chromatin at the mating-type locus. This RNAi-directed form of centromeric heterochromatin provides an ideal system for in vivo screening to allow the identification of compounds that inhibit the activity of proteins involved in RNA silencing, chromatin modification and heterochromatin assembly in fission yeast and may inhibit conserved proteins in other organisms. A dominant selectable marker gene system at fission yeast centromeres that reports loss of heterochromatin integrity by increased resistance to G418 in 96-well plate format liquid cultures was developed. The resulting strain was used to screen a nontargeted chemically diverse compound library in vivo to identify compounds that disrupt the integrity of RNAi-directed heterochromatin. Two compounds, Emi1 and Emi14, were identified and found to cause a significant decrease in the level of H3K9 methylation on the outer repeats at fission yeast centromeres. Growth in the presence of Emi1 or Emi14 also caused a reduction in H3K9 methylation levels at the mating-type locus, suggesting that they do not act through RNAi. Consistent with this, Emi1 and Emi14 did not cause a decrease in centromeric siRNA levels. Analyses therefore suggest that Emi1 and Emi14 do not disrupt RNAi but that they inhibit downstream events in chromatin modification and heterochromatin assembly. Cells lacking RNAi due to loss of Dicer (dcr1Δ) or cells lacking the histone deacetylase (HDAC) Sir2 (sir2Δ) retain significant but lower levels of H3K9 methylation on the centromeric outer repeats. When dcr1Δ or sir2Δ cells were grown in the presence of Emi1 or Emi14 a further reduction in H3K9 methylation levels was observed on the outer repeats. This mimics the effect of combining clr3Δ with dcr1Δ or sir2Δ and suggests that Emi1 and Emi14 may interfere with SHREC function. SHREC is a chromatin remodelling complex that includes the HDAC Clr3 and the chromatin remodeler Mit1 and is known to contribute to heterochromatin integrity. Expression profiling performed on Emi1 and Emi14 treated cells confirmed the previous results. The changes in gene expression following Emi1 and Emi14 treatment were compared to known mutants defective in heterochromatin integrity. The profile of expression changes following Emi14 treatment was found to correlate with alterations in the expression pattern observed in cells with SHREC components deleted. No correlation with mutants lacking other HDACs or RNAi components was detected. Emi1 had a weaker correlation with defective SHREC function and thus may also partially inhibit the SHREC complex. Murine erythroleukemia (MEL) cells harbouring a silenced eGFP reporter transgene were used to assess whether Emi1 and Emi14 also affect silencing in mammalian cells. Emi1 was found to disrupt silencing at the eGFP reporter and this correlated with a decrease in H3K9 methylation. Structurally related analogues of Emi1 and Emi14 were selected and tested in the fission yeast assay. Interpretation of the obtained structure-activity relationships allowed identification of the chemical moieties key to Emi1 and Emi14 activity. Overall, an approach was developed to identify two novel small molecule inhibitors of a well-characterized chromatin modification pathway. The SHREC complex was identified as the putative target of these two compounds and structurally related active analogues were identified for them. Importantly, one of the compounds was also active in mammalian cells, highlighting the usefulness of this approach in identifying compounds that affect higher organisms.

This dissertation describes our study of a photophysical process that leads to ultrafast generation of triplet excitons following photoexcitation, singlet exciton fission, in three different acene dimers and diradicaloids. In pentacene and tetracene dimers, we investigate their mechanism of singlet fission. In a series of diradicaloids, we study the relation between molecular structure, diradical character and the suitability for singlet fission. In the first two chapters we explore singlet fission in pentacene dimer. We demonstrate fast and highly efficient intramolecular singlet fission, consisting of two covalently attached pentacene units. The singlet fission pathway is governed by the energy gap between singlet and charge-transfer states, which change dynamically with molecular geometry but are primarily set by the side group. The process exhibits a sensitivity to solvent polarity and competes with geometric relaxation in the singlet state, while subsequent triplet decay is strongly dependent on conformational freedom. The near orthogonal arrangement of the pentacene units is unlike any structure currently proposed for efficient singlet fission and points toward new molecular design rules. Furthermore, these results are the first to demonstrate the role of charge-transfer states in singlet fission and highlight the importance of solubilising groups to optimise excited-state photophysics. In the next chapter, we examine singlet fission in tetracene dimer, where singlet fission is energetically unfavourable. We demonstrate triplet yield as high as 190% can be achieved via fission from higher singlet excited states mediated by charge-transfer states. The outcomes of this study provide deeper insight into the role of hot singlet states in singlet fission and point toward less stringent molecular design rules. In the last chapter, we shift our focus on a new class of molecules, diradicaloid molecules. We explore a family of zethrene molecules, with tuneable diradical character, and demonstrate their general ability to undergo rapid singlet fission via spin-entangled and emissive triplet-pair state TT. A wide range of zethrene molecules are found to be suitable for singlet fission, with additional benefits of high absorption coefficients and photo-/chemical stability.

Foi desenvolvido, implementado e testado um formalismo que permite descrever propriedades nucleares em regimes de deformação fora do equilíbrio, utilizando o método Macroscópico-Microscópico em associação com o Método Semimicroscópico Combinado. Esse formalismo foi utilizado para a interpretação e elucidação de alguns problemas abertos como as estruturas na seção de choque de fotofissão do 237.ANTPOT Np, distribuições angulares de fotofissão do 239.ANTPOT. Pu, e a concentração de transições M1 no 237.ANTPOT Np e 239.ANTPOT. Pu em baixas energias. Também, foram interpretadas informações experimentais referentes aos estados isoméricos do 237.ANTPOT Pu e 239.ANTPOT. Pu. Com relação ao potencial preditivo do formalismo, foram identificados os possíveis candidatos (spin e paridade) a estados isoméricos no 241.ANTPOT Np, bem como foi calculada também pela primeira vez a distribuição de momentos de nêutrons para o 239.ANTPOT Np fora da deformação de equilíbrio. / It was developed, implemented and tested a formalism suitable to the description of nuclear properties at non equilibrium deformations regime, by using the Macroscopic-Microscopic method in association with the Combined Semimicroscopic Method. This formalism was used for the interpretation and elucidation of some open problems, as the structures in the 237Np photofission cross section, photofission angular distributions of 239Pu, and the concentrations of low-energy Ml transitions in 237Np and 239Pu. Also, experimental information on 237Pu and 239Pu isomeric states was interpreted. Concerning the prediction potentialities of the formalism, were identified possible candidates (spin and parity) for the 241Pu isomeric states, as well as the 239Pu neutron momentum distributions, at non equilibrium deformation, was also calculated for the first time.

Following a NEA high priority nuclear data request, an experimental campaign to measure the prompt Î3-ray emissions from 235 U has been performed. This has used the STEFF spectrometer at the new Experimental Area 2 (EAR2) within the neutron time- of-flight facility (n_TOF), a white neutron source facility at CERN with energies from thermal to approximately 1 GeV. Prior to the experimental campaign, STEFF has been optimised for the environment of EAR2. The experimental hall features a high background Î3-ray rate, due to the nature of the spallation neutron source. Thus an investigation into reduction of the background Î3-ray rate, encountered by the NaI(Tl) detector array of STEFF, has been carried out. This has been via simulations using the simulation package FLUKA. Various materials and shielding geometries have been investigated but the effects determined to be insufficient in reducing the background rate by a meaningful amount. The NaI(Tl) detectors have been modified to improve their performance in a high count rate environment, and their behaviour characterised to understand the response to higher count rates. Initial testing demonstrated that the modified detectors maintain a potential to measure Î3-ray multiplicities up to 3 counts per microsecond. However, the energy resolution fails somewhere below 1.75 counts per microsecond. The experimental campaign has produced a large amount of data. The preliminary analysis of phase one data has considered incoming neutron energies ranging from thermal to an upper limit of 1 eV, with a minimum Î3-ray energy threshold of 160 keV. Results have been achieved for the prompt fission Î3-ray multiplicity and total energy of M Î3 = 6.3 ± 0.2 and E S,Î3 = 9.0 ± 0.1 MeV respectively. Further work is ongoing by the STEFF team at Manchester to improve upon these results and analyse the remainder of the data set at higher incoming neutron energies.

Mto1 recruits γ-tubulin to the sites of cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe. The regulation of Mto1 localisation is central to re-modelling of the microtubule cytoskeleton during the cell cycle. This thesis describes how Mto1 is recruited to the cell equator during mitosis, thereby establishing the equatorial microtubule nucleation centre (eMTOC). F-actin is found to be required for Mto1 localisation to the cell equator and Mto1 is shown to co-localise with the cytokinetic actin ring (CAR). Yeast 2-hybrid screening and tandem-affinity purification were used to screen for additional proteins required for Mto1 localisation to the equator. Further candidate screening identified Myp2, a type II myosin present in the CAR, as being required for Mto1 localisation to the cell equator. Myp2 is shown to physically interact with Mto1 and to be required for formation of the post-anaphase microtubule array. The regulation of Mto1 localisation to the cell equator was also studied. Time-lapse microscopy reveals that Mto1 localisation to the equator does not require either the anaphase-promoting complex or the septation initiation network, both of which have been previously shown to be necessary for the recruitment of γ-tubulin to the eMTOC. Maintenance of the equatorial CAR has previously been attributed to the postanaphase array. The position of the CAR in the mto1-427 mutant strain, which fails to nucleate a PAA, is shown to be unaltered from wild-type strain during exponential growth, suggesting that the PAA does not centre the CAR during normal growth.

Spin interactions play a key role in the function of molecular materials from naturally occurring biological complexes to synthetic materials for light-harvesting and light-emission. This thesis investigates the spin interactions of spin-1 triplet excitons formed by singlet fission. Singlet fission produces two triplet excitons from one light-induced singlet state and holds promise to enable solar energy generation beyond traditional efficiency limits. As the lifetime of triplet pairs depends sensitively on their spin degree of freedom, in this thesis we deploy spin-sensitive techniques to understand the interactions and evolution of triplet pairs. After introducing the relevant theoretical and experimental background underlying singlet fission and the role of spin, we describe the first observation of strongly exchange coupled, high-spin triplet-pair states ($S=2$) in a solid-state organic semiconductor and show that the singlet fission process allows for the formation of long-lived, strongly coupled spin-two states. We then describe the development and use of photoluminescence-detected avoided level-crossings in applied magnetic fields to quantify the strength of exchange coupling and identify specific optical signatures of exchange-coupled triplet pairs. Using high magnetic fields ($\leq\mbox{60 T}$) we isolate and measure the exchange coupling and optical signatures of multiple distinct triplet pairs in the same material. Finally, we probe the mechanisms of formation and decay of spin polarization from triplet pair states using pulsed spin resonance. The measured dynamics are consistent with polarization driven by fluctuations in exchange coupling between pairs and spin-orbit mediated decay of triplet excitons to the ground state. The combined measurements of the spin parameters and polarization dynamics of triplet pairs from ns to ms timescales provides a quantitative picture of the spin states generated by singlet fission.

O Brasil planeja construir uma planta de produção do 99Mo por fissão do 235U, devido à crescente utilização deste radioisótopo no setor de medicina nuclear. Neste trabalho buscou-se estimar as características físico-químicas e radiológicas dos rejeitos radioativos que serão gerados em tal instalação, além de fornecer subsídios teóricos a serem utilizados na definição do gerenciamento desses rejeitos. Estabelecidos dois cenários de produção para este projeto e utilizando-se o código Scale®, foram calculados os inventários radioisotópicos dos fluxos de rejeitos. Considerando-se o processamento químico dos alvos de urânio, foi possível caracterizar os rejeitos a partir de suas características químicas e radiológicas. Utilizando-se o software MicroShield®, determinou-se então as concentrações de atividade desses rejeitos em até 3 meses de produção. Este trabalho ainda apresenta o cálculo das taxas de dose variando-se tanto a espessura da blindagem, a ser utilizada numa embalagem para transporte in-site, quanto a quantidade de rejeito líquido retido em tal embalagem. Os radionuclídeos responsáveis pela maior parcela da dose de radiação foram identificados de forma a facilitar a determinação do método mais apropriado para o gerenciamento dos rejeitos após sua separação e antes de seu armazenamento. Os resultados aqui apresentados constituem uma compilação inicial do que é esperado em termos de rejeitos radioativos líquidos numa planta produtora de 99Mo e podem auxiliar no desenvolvimento do plano de gerenciamento de rejeitos de tal instalação. / Brazil intends to build a facility for the 99Mo production through 235U fission, once this radioisotope is largely used in nuclear medicine. This study aimed at estimating the physical, chemical and radiological characteristics of radioactive waste expected to be generated in that facility, and to provide theoretical subsides that can be used on the definition of a proper waste management system. Two production scenarios were established and the radioisotope inventories of the wastes were calculated by Scale®. From the chemical processing of the uranium targets the wastes were characterized on their chemical and radiological features. MicroShield® was used to determine the activity concentrations up to three months of 99Mo production. In addition, this work presents dose rate calculation for several sizes of shielding and different amount of wastes, collected in a proper package for in-site transportation. Radionuclides responsible for higher doses were identified in order to facilitate choosing the most appropriate method for managing the wastes after their chemical separation and before their storage. These results are part of what is expected on radioactive wastes at a 99Mo production facility and might help on the development of the waste management planning for that facility.

Serine/threonine phosphatase 2A (PP2A) consists of an AC core dimer composed of catalytic (C), structural (A) subunits complexed to a variable regulatory subunit derived from three gene families (B, B', B"). My dissertation work characterized the structure and function of a neuron-specific splice variant of the Bbeta regulatory gene termed Bbeta2. I found that the divergent N-terminus of Bbeta2 does not affect phosphatase activity or holoenzyme association but encodes a mitochondrial targeting signal. Moreover, transient and stable expression of wild-type Bbeta2 but not Bbeta1, Bbeta2 mutants defective in mitochondrial targeting or a monomeric mutant unable to associate with the holoenzyme, promotes apoptosis in neurons while knock-down of endogenous Bbeta2 is neuroprotective. Furthermore, I identified the mechanisms by which Bbeta2 incorporates the PP2A holoenzyme. By performing charge reversal mutagenesis in Bgamma as a model for B family regulatory subunits, I found that holoenzyme association requires multiple electrostatic charges clustered in WD repeats 3 and 4 of the beta-propeller. To identify residues in Bbeta2 important for mitochondrial association, I performed mutagenesis of the divergent N-terminus of Bbeta2 and identified basic and hydrophobic residues that are critical for mitochondrial association. The variable N-terminal tail of Bbeta2 is a cryptic mitochondrial import sequence that promotes import of GFP, but not full-length Bbeta2, because its beta-propeller domain resists the partial unfolding step necessary for translocation. Lastly, I addressed the mechanism by which Bbeta2 promotes apoptosis in neurons. I found that overexpressing Bbeta2 fragments mitochondria while RNAi of the endogenous protein promotes mitochondrial fusion in neurons. Conversely, targeting PKA, a well characterized prosurvival kinase, to the OMM by overexpressing A kinase anchoring protein 121 (AKAP121) opposes the effects of the phosphatase by elongating mitochondria. Furthermore, downregulating the endogenous AKAP121 by RNAi, or inhibiting PKA at the OMM by overexpressing an inhibitor of PKA (OMM-PKI) fragments mitochondria. The effects of OMM-targeted PP2A or PKA on survival require remodeling of mitochondria, since blocking mitochondrial fission reversed the proapoptotic effects of Bbeta2 and OMM-PKI. My dissertation provides a novel mechanism by which kinase/phosphatase signaling determines neuronal survival.

Mitochondria are dynamic organelles that constantly undergo opposing fission and fusion events which impact many aspects of mitochondrial and cellular homeostasis including bioenergetic activity, calcium buffering and organelle transport. The large GTPase dynamin-related protein 1 (Drp1) acts as a mechanoenzyme to catalyze fission of mitochondria. Drp1 activity is regulated through a series of reversible posttranslational modifications. Phosphorylation of the conserved serine residue, S656, by cAMP dependent protein kinase A (PKA) acts as a master regulator of Drp1 activity. Two phosphatases oppose PKA by dephosporylating Drp1 S656, a mitochondrial isoform of protein phosphatase 2A and the calcium-calmodulin dependent phosphatase calcineurin (CaN). Here I report the characterization of a conserved CaN docking site on Drp1, an LxVP motif, just upstream of the Drp1 S656 site. Mutational modification of the Drp1 LxVP motif resulted in selective bidirectional modulation of formation of the CaN:Drp1 complex. Stability of the CaN:Drp1 LxVP motif mutant complexes was qualitatively described by affinity purification and quantitatively described by isothermal titration calorimetry. Stability of the CaN:Drp1 complex was found to directly correlate with Drp1 S656 dephosphorylation kinetics as demonstrated by studies conducted in vitro and in intact cells. Further, the CaN:Drp1 signaling axis was shown to shape basal mitochondrial morphology in a heterologous cell line system and in primary hippocampal neurons. Finally, disruption of the CaN:Drp1 signaling axis was found to protect neurons from oxygen-glucose deprivation, an in vitro model of ischemic injury. While these results suggest that the CaN:Drp1 signaling axis may be a potential target for neuroprotective therapeutic exploitation, the mechanism by which disruption of the CaN:Drp1 signaling axis specifically and mitochondrial elongation generally results in resistance to ischemic injury remains unknown. Additional studies reported here demonstrate that mitochondrial fragmentation remains a prominent feature of injured neurons regardless of the fidelity of the CaN:Drp1 signaling axis. Mitochondrial fragmentation at the time of injury was found to occur in a Drp1-independent manner. Chronic mitochondrial elongation was also found to leave unaltered the ability of neurons to detoxify reactive oxygen species, buffer intracellular calcium and supply ATP for homeostatic function.

Eukaryotic cells are unique in their ability to form complex multicellular organisms giving rise to distinct physiological systems. However, the ability for such complexity to evolve likely stems from an early event in which endosymbiosis of an aerobic prokaryote by a eukaryotic precursor gave rise to the eukaryotic organelle we now know as mitochondria. Mitochondria are colloquially known as the “power house” of the cell due to their ability to produce ATP through oxidative phosphorylation, but perform numerous other vital functions within the cell including sequestration of cytosolic Ca2+, production and sequestration of reactive oxygen species (ROS), and initiation of various forms of cell death. Mitochondria are especially important in neurons given their high demand for ATP and the importance of Ca2+ signaling in neuron excitability and development. Neurons are highly compartmentalized and plastic cells requiring the ability to control energy supply and Ca2+ signaling locally within given specialized structures such as dendritic spines or synaptic boutons. Therefore, mitochondria must be able to localize to particular sub-cellular locales and respond functionally to signaling occurring in that environment. Mitochondrial transport and function are heavily dependent upon the ability of mitochondria to undergo opposing and reversible fission and fusion events. Mitochondrial fission and fusion are themselves regulated by GTPase enzymes which physically catalyze constriction and fusion of the mitochondrial membranes. Mutations in mitochondrial fission and fusion enzymes specifically cause neurological disease in humans and recent work has illustrated the necessity of a proper balance of mitochondrial fission in neuron development, survival, and plasticity. Despite recognizing the importance of mitochondrial fission and fusion in neuron survival, development, and function we lack a concrete understanding of how changes in the equilibrium of fission and fusion impact these processes in vivo. In this thesis we investigate how promoting or inhibiting mitochondrial fission, through phosphoregulation of the mitochondrial fission enzyme Dynamin related protein 1 (Drp1) at mitochondria, impacts neuron survival and memory in vivo. We find that inhibiting phosphorylation of Drp1 at Serine 656 (S656) at the mitochondria, through deletion of a mitochondrial targeted A kinase anchoring protein (AKAP) known as AKAP1 in mice, increases cerebral infarct volume following transient occlusion of the mid-cerebral artery. Oppositely, promoting phosphorylation of Drp1-S656 at the mitochondria, through deletion of the PP2A regulatory subunit Bβ2 which localizes the PP2A heterotrimer to mitochondria, decreases cerebral infarct volume following occlusion of the mid-cerebral artery. Mechanistic in vitro studies in primary neurons reveal these effects are dependent upon the phosphorylation state of Drp1-S656 and likely due to altered mitochondrial respiratory capacity, ROS production, and Ca2+ homeostasis. Interestingly, we also observe improved hippocampal dependent memory in mice in which AKAP1 has been deleted which also appears dependent upon the phosphorylation state of Drp1-S656 and Ca2+ homeostasis. Ultimately, these findings provide insight into how phosphoregulation of Drp1 at the mitochondria alters neuron survival and function through shifting the mitochondrial fission/fusion equilibrium and consequently mitochondrial function.