<p>Conserved across metazoans, cell division depends upon the synchronous assembly and disassembly of a robust, mitotic spindle for the congression and separation of duplicated chromosomes. Composed of mostly microtubules, mitotic spindle generation depends on three different microtubule nucleation mechanisms to build its distinctive bipolar assembly. These three mechanisms are centrosomal-based, kinetochore-based, and branching microtubule nucleation. Branching microtubule nucleation occurs when microtubules nucleate from the sides of pre-existing microtubules within the mitotic spindle. Without branching microtubules, a weaker spindle apparatus can result in mitotic delay, chromosomal misalignment, multi-polar spindles, and/or aneuploidy. </p> <p>Several important complexes and proteins mediate branching microtubule nucleation. These proteins are the γ-tubulin ring complex (γ–TuRC), the homologous to augmin subunits (HAUS) complex (or simply augmin), the targeting protein for Xklp2 (TPX2), colonic and hepatic tumor overexpressed gene (chTOG), and echinoderm microtubule-associated protein-like 3 (EML3) among others. This work focused on discerning the molecular architecture of the augmin complex while also endeavoring to establish heterologous expression and purification methodologies for the γ–TuRC and TPX2. </p> <p>Augmin consists of proteins HAUS1–8 (H1–8) which bind to the sides of pre-existing microtubules and orient the γ–TuRC, the template for making microtubules, via NEDD1 to create new microtubules at shallow angles (~<20°). Despite its importance in cell division, the structure of augmin has eluded determination. This work utilized a multi-pronged approach of the baculovirus insect cell protein complex expression, cryo-EM, new protein structure prediction methodologies, and crosslinking mass spectrometry (CLMS) to elucidate the molecular architecture of the augmin complex. Further work studying the isolation, structure prediction and comparison across model organisms, and phosphorylation studies was also conducted. The results will aid the structure-assisted development of novel chemotherapeutics that target the augmin complex as well as provide deeper insights into how this complex functions in cell division. </p> <p>To help better understand the molecular mechanisms, regulation, and interactions between the different machinery involved in branching microtubule nucleation, the γ–TuRC and TPX2 also became a focus of this work. My primary effort was to overexpress and purify from the heterologous baculovirus insect cell protein complex expression system sufficient quantities of γ–TuRC for biochemical and biophysical characterization. Thus, efforts shifted to establish an expression and purification methodology for this complex. Similarly, a methodology for purification of TPX2 were also initiated. The goal of these endeavors is to establish <em>in vitro</em> biochemical reconstitution of branching microtubule nucleation utilizing the augmin complex, γ–TuRC, and TPX2 utilizing total internal reflection fluorescence microscopy (TIRF-M). </p> <p>Lastly, in unrelated work, a section on other work focuses on the roles of anti-CRISPR proteins that inhibit the Csy surveillance complex from <em>Pseudomonas aeruginosa</em> can be found. Cryo-EM studies revealed the structures of AcrIF4, AcrIF7, and AcrIF14. These anti-CRISPR proteins inhibit the Csy complex by different mechanisms. AcrIF4 prevents conformational changes necessary to recruit a Cas2/3 nuclease for degradation of invading mobile genetic elements while AcrIF7 acts as a dsDNA mimic preventing invading phage DNA recognition. Lastly, AcrIF14 functions by binding in the grove where the crRNA of Csy is and prevents hybridization between target invading MGE DNA and the crRNA. These mechanisms exemplify convergent evolution among anti-CRISPR proteins while also showing the diversity of structures produced by phages in their ongoing molecular arms race with their hosts.</p>

Cardiac diseases such as myocardial infarction (MI) can lead to adverse remodeling and impaired contractility of the heart due to widespread cardiomyocyte death in the damaged area. Current therapies focus on improving heart contractility and minimizing fibrosis with modest cardiac regeneration, but MI patients can still progress to heart failure (HF). There is a dire need for clinical therapies that can replace the lost myocardium, specifically by the induction of new myocyte formation from pre-existing cardiomyocytes. Many studies have shown terminally differentiated myocytes can re-enter the cell cycle and divide through manipulations of the cardiomyocyte cell cycle, signaling pathways, endogenous genes, and environmental factors. However, these approaches result in minimal myocyte renewal or cardiomegaly due to hyperactivation of cardiomyocyte proliferation. Finding the optimal treatment that will replenish cardiomyocyte numbers without causing tumorigenesis is a major challenge in the field. Another controversy is the inability to clearly define cardiomyocyte division versus myocyte DNA synthesis due to limited methods. A recent study suggests that systemic hypoxemia in adult male mice can induce cardiac myocytes to proliferate. The goal of the present experiments was to confirm these results, provide new insights on the mechanisms that induce cardiomyocyte cell cycle re-entry, and to determine if hypoxemia also induces cardiomyocyte proliferation and division in female mice. EdU mini pumps were implanted in 3-month-old, male and female C57BL/6 mice. Mice were then placed in a hypoxia chamber and the oxygen was lowered by 1% every day for 14 days to reach 7% oxygen. The animals remained in 7% inspired oxygen for 2 weeks before terminal studies. Myocyte cell cycle re-entry and division was also studied with a mosaic analysis with double markers (MADM) mouse model. MADM mice were exposed to hypoxia at 7% Oxygen as described above. Hypoxia induced cardiac hypertrophy in both left ventricular (LV) and right ventricular (RV) myocytes, with LV myocytes lengthening and RV myocytes widening and lengthening. Hypoxia induced a small increase in cardiomyocytes undergoing DNA synthesis (EdU+) in male and female C57BL/6 mice. Hypoxia induced a significant increase in myocyte cell cycle re-entry in MADM mice, but few myocytes synthesized new DNA (EdU+) and completed cytokinesis. RNA-sequencing showed upregulation in mitotic cell cycle processes but a downregulation of promoter genes for G1 to S phase transition in hypoxic mice when compared to control mice. There was also proliferation of non-myocyte cells and mild cardiac remodeling in hypoxic mice that did not disrupt cardiac function. Male and female mice exhibited similar gene expression profiles following hypoxia. Thus, systemic hypoxia induces adult cardiac myocyte cell cycle re-entry, but very few adult myocytes progress through the cell cycle to synthesize new DNA and divide into two daughter cells. / Biomedical Sciences

Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. The streptomycetes divisome is however, similar to that of other prokaryotes. We hypothesized chemical inhibitors of sporulation in model streptomycetes might interfere with cell division in rod shaped bacteria. To test this, we investigated 196 compounds that inhibit sporulation in Streptomyces coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, 2 and 3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but which is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors. In addition to the molecules affecting the divisome, DNA and cell wall damage also affects the process indirectly by temporarily halting the cell division. To further explore the cell division regulation in stressful conditions, we carried the complete transcriptomic analysis of S. venezuelae after the DNA damage. The observed changes in the gene expression as a result of the DNA damage paves the way for identification of the DNAdamage induced cell division inhibitor in streptomycetes. / Thesis / Doctor of Philosophy (PhD)

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No / The aim of this study was to develop a synthetic hydrogel to act as a corneal substitute capable of selectively supporting the adhesion and proliferation of limbal epithelial cells (LECs) while inhibiting growth of limbal fibroblasts. Deficiency of LECs causes conjunctival epithelial cells to move over the cornea, producing a thick scar pannus. Unilateral defects can be treated using LEC cultured from the unaffected eye, transplanting them to the affected cornea after scar tissue is removed. The underlying wound bed is often damaged, however, hence the need to develop a corneal inlay to aid in corneal re-epithelialization. Transparent epoxy-functional polymethacrylate networks were synthesized using a combination of glycerol monomethacrylate, ethylene glycol dimethacrylate, lauryl methacrylate and glycidyl methacrylate that produced two different bulk hydrogel compositions with different equilibrium water contents (EWCs): Base 1 and Base 2, EWC=55% and 35%, respectively. Two sets of amine-functional hydrogels were produced following reaction of the epoxide groups with excesses of either ammonia, 1,2-diamino ethane, 1,3-diamino propane, 1,4-diamino butane or 1,6-diamino hexane. Neither series of hydrogels supported the proliferation of limbal fibroblasts irrespective of amine functionalization but they both supported the adhesion and proliferation of limbal epithelial cells, particularly when functionalized with 1,4-diamino butane. With Base 1 hydrogels (less so with Base 2) a vigorous epithelial outgrowth was seen from small limbal explants and a confluent epithelial layer was achieved in vitro within 6days. The data support the development of hydrogels specific for epithelial formation.

La transformation de la paroi de l’ovaire en un péricarpe charnu implique une coordination entre les divisions cellulaires et l’expansion cellulaire. Des données considérables sur le développement et la maturation du fruit de tomate ont été établies, mais la coordination des divisions cellulaires, de l’expansion cellulaire et de l’endoréduplication durant la mise à fruit ainsi que durant la croissance du fruit de tomate reste grossièrement caractérisée au sein du péricarpe et de nombreuses questions ne sont pas résolues : comment ces deux processus sont-ils régulés et coordonnés durant le développement du fruit d’un point de vue cellulaire? Quand commence l’endoréduplication dans les tissus du fruit et quelle est sa fonction? La première partie de ce mémoire concerne la coordination des divisions cellulaires et de l’expansion cellulaire durant la fin du développement de l’ovaire et le début du développement du fruit. Une différenciation précoce des assises cellulaires composant la paroi de l’ovaire puis le péricarpe a été démontrée. Les divisions cellulaires se font principalement au sein de l’épiderme externe et montrent une synchronisation partielle tandis que l’expansion cellulaire se fait principalement dans le mésocarpe. L’endoréduplication semble être initiée avant l’anthèse. La deuxième partie est consacrée à l’analyse du RNA-seq nucléaire en fonction de quatre niveaux de ploïdie (4, 8, 16 et 32C). La majorité des gènes montrent une augmentation proportionnelle de leurs expressions en fonction des niveaux de ploïdie. Cependant, certains gènes révèlent une surexpression ou une sous-expression en fonction des niveaux de ploïdies. / The transformation of the ovary wall into a fleshy pericarp involves a coordinated pattern of cell division and cell expansion. Considerable data have been reported on tomato fruit development and ripening, but the pattern of cell division, cell expansion and endoreduplication at the tomato fruit set and during fruit growth remains grossly appreciated at the whole pericarp level and many questions are not yet resolved: How are cell division and cell expansion coordinated in tomato fruit a cellular level and according to developmental time? When does endoreduplication begin in fruit tissues and what is its function? The first part of this deals with the coordination of cell division and cell expansion during the end of tomato ovary development and the beginning of fruit growth. Evidence for early differentiation of cell layers in the ovary wall and then in fruit pericarp are presented. Cell division happens mainly in the external epidermis and shows partial synchronization, whereas cell expansion happens mostly in mesocarp cell layers. Endoreduplication is initiated as soon as before anthesis. The second part of this work is devoted to RNA-seq based transcriptome profiling of pericarp nuclei which have been sorted according to four ploidy levels (4, 8, 16 and 32C). We demonstrate that the expression of most of the pericarp-expressed genes shows a proportional increase according to ploidy level, on a nuclear basis. However, a significant amount of genes has been identified as over-expressed or under-expressed according to ploidy level.

A divisão celular em B. subtilis inicia-se pela formação de um complexo multiprotéico, o divisomo, no sítio onde a bactéria irá se dividir. FtsZ é a primeira proteína a se localizar no futuro sitio de divisão, formando uma estrutura em anel (anel Z) que se estende por toda a circunferência da célula. O anel Z funciona como um arcabouço responsável por recrutar outras quinze proteínas de divisão que irão participar da montagem do divisomo. Nesta tese, utilizamos abordagens quantitativas e qualitativas de microscopia de fluorescência vital para estudarmos duas questões ainda não esclarecidas sobre o funcionamento do divisomo. A primeira delas é como o divisomo é montado. Para estudarmos a montagem do divisomo nós realizamos ensaios de co-localização entre o anel Z (FtsZ-mCherry) e as proteínas ZapA, EzrA, FtsW, FtsL, YpsB , DivIVA, e MinC fusionadas a GFP. Quanto maior a freqüência de co-localização entre FtsZ e outra proteína de divisão, mais inicial é a participação da proteína na formação do divisomo. Portanto, a medida da freqüência de co-localização entre o anel Z e as proteínas componentes do divisomo permite que se deduza uma cinética da montagem deste complexo. Estes ensaios demonstraram uma freqüência de co-localização de 97,33% para ZapA; 98,31% para EzrA; 83,90% para FtsW; 78,43% para FtsL; 50% para YpsB; 41,7% para DivIVA e 31,64% para MinC. Estes resultados sugerem que o divisomo seja formado em três etapas. ZapA e EzrA se associam ao divisomo imediatamente após a formação do anel Z, em seguida FtsW e FtsL são recrutados para o divisomo, e por último YpsB, DivIVA, MinC associam-se ao divisomo. A segunda questão que investigamos nesta tese foi o mecanismo da mudança de posição do divisomo que ocorre durante a esporulação em B. subtilis. Na fase de esporulação a célula divide-se assimetricamente, com a formação do septo próxima a um dos pólos. Durante o crescimento vegetativo a divisão não ocorre próxima aos pólos por causa da ação das proteínas MinC, MinD e DivIVA, importantes reguladores espaciais da divisão. MinCD e DivIVA são inibidores da formação do anel Z que durante o crescimento vegetativo se localizam nos pólos das células.. Uma hipótese para explicar o uso dos sítios polares para a divisão durante a esporulação seria que as proteínas MinCD e DivIVA seriam removidas dos pólos celulares. Para testarmos esta hipótese, estudamos a localização das proteínas MinCD e DivIVA durante a esporulação. Nossos resultados demonstraram que MinCD e DivIVA se re-localizam e saem dos pólos celulares durante a esporulação. Porém esta dinâmica ocorre após a formação do anel Z assimétrico, sugerindo que o anel Z seja insensível a estes inibidores durante a esporulação. Por ensaios genéticos em B. subtilis demonstramos que a proteína SpoIIE, conhecida como provável proteína responsável por promover a formação do septo assimétrico, seja capaz de contrapor a ação de MinC no início da esporulação. Dessa maneira nós propomos um novo modelo de mudança da divisão simétrica para assimétrica durante a esporulação, diferentemente da simples saída do complexo MinCD dos pólos como é proposto na literatura. / Bacillus subtilis division begins through the formation of a multiprotein complex, the divisome, at the site of division. FtsZ is the earliest known protein to localize to the future division site where the protein forms a ring-like structure (Z-ring) that extends around the circumference of the cell. The Z-ring functions as a scaffold and recruits about fifteen other division proteins that compose the divisome. In this work, we used quantitative and qualitative methods of vital fluorescence microscopy to study two questions that have not been elucidated about the divisome dynamics. The first is how divisome is assembled. To address that problem, we made co-localization between Z-ring (FtsZ-mCherry) and proteins ZapA, EzrA, FtsW, FtsL, YpsB, DivIVA, and MinC fused to GFP. Higher is the match between GFP fusions to Z-ring, earlier is the assembly of division proteins to divisome. Therefore, the co-localization frequency between Z ring and divisome proteins will allow us to deduce the assemble kinetics of the divisome. This assays showed a co-localization frequency of 97,33% for ZapA; 98,31% for EzrA; 83,90 for FtsW; 78,43% for FtsL; 50% for YpsB; 41,7% for DivIVA and 31,64% for MinC. This data suggests that the divisome does not assemble in two but in three steps. ZapA and EzrA assemble into the divisome immediately after Z ring formation, secondly FtsW and FtsL were recruited to the divisome, and finally YpsB, DivIVA, MinC associated with the divisome. The second question that we investigated in this work is the mechanism responsible for change the divisome position that occurs during sporulation in B. subtilis. In sporulation the cell divides asymmetrically, with a septum formation near poles. During vegetative grown the divisiome does not occur near poles because of MinC, MinD and DivIVA action, relevant for spatial regulation of division. MinCD and DivIVA are inhibitors of Z ring formation that during vegetative growth are located at poles. A hypothesis to explain the use of polar sites for division during sporulation would be that MinCD and DivIVA would be removed from cellular poles. To test this hypothesis, we studied the location of MinCD and DivIVA proteins during sporulation. Our results demonstrated that MinCD and DivIVA re-localize and leave to cell poles during sporulation. However this process occurs after asymmetric Z ring formation, suggesting that Z ring would be unresponsive to this inhibitors during sporulation. Through genetics assays in B. subtilis we demonstrated that SpoIIE protein, known to probably play a role in asymmetric septum formation, would be able to contrapose MinC action during early sporulation. Therefore, we propose a novel model for change the symmetric to asymmetric division during sporulation, unlike the release of MinCD from pole proposed in the literature.

A utilização do vírus adeno-associado recombinante (AAVr) como vetor de transferência gênica em células tumorais está crescendo. Neste trabalho, o promotor gênico de E2F-1, um promotor ativo durante a divisão celular, foi inserido no AAVr e utilizado para dirigir a expressão do HSV-tk ou luciferase e, simultaneamente, eGFP afim de direcionar a expressão viral para células em proliferação. Em paralelo, foram construídos vetores portadores do promotor constitutivo CMV para servir como controles. O promotor gênico de E2F-1 não foi eficiente em dirigir a expressão dos transgenes na linhagem celular HT1080, enquanto o promotor CMV apresentou uma alta expressão dos repórteres e do gene terapêutico. A baixa eficiência do promotor E2F-1 ainda não foi explorada, mas poderia ser relacionada com o desempenho intrínseco deste promotor, a biologia do vetor AAVr e especificidade celular. Contudo, o bom desempenho do vetor AAVr contendo o promotor CMV abre a possibilidade de realizar novos ensaios de transferência gênica para tratamento e visualização de células tumorais / The utilization of recombinant adeno-associated virus (AAVr) as a gene transfer vector in tumor cells is increasing. In this work, the promoter of the E2F-1 gene, active during cell division, was inserted in an AAVr vector and used to drive the expression of HSV-tk or luciferase and, simultaneously, eGFP with the intent of limiting viral expression to proliferating cells. Also, vectors with the constitutive CMV promoter were constructed to be used as controls. The E2F-1 promoter was not efficient in driving the expression of the transgenes in the HT1080 cell line, while the CMV promoter shows high level expression of the reporter and the therapeutic genes. The low efficiency of E2F promoter has not yet been explored, though this problem could be related to the intrinsic performance of this promoter, the biology of the vector AAV and cell-specific factors. However, the performance of the AAVr containing the CMV promoter creates the possibility of performing new gene transfer protocols for the treatment and visualization of tumor cells.