In fission yeast, microtubule (MT) nucleation is regulated in space and time. In interphase, MTs are nucleated in the cytoplasm to regulate cell polarity, whereas in mitosis, nucleation takes place inside the nucleus to form a mitotic spindle. We hypothesize that several non-exclusive mechanisms may be responsible for this differential regulation of MT nucleation. Two fission yeast proteins, Mto1 and Pcp1, are involved in MT nucleation in interphase and mitosis, respectively. These proteins share a sequence motif, called CM1 that is responsible for interaction with the γ-tubulin complex (γ-TuC). In the first part of my project, I tested whether sequence differences between Mto1 and Pcp1 CM1 regions contribute to the differential regulation of MT nucleation in interphase vs. mitosis. I showed that the two CM1 regions are interchangeable and play no role in differential regulation of Mto1 and Pcp1. By generating Pcp1-9A1 mutant, where conserved residues within the Pcp1 CM1 region was replaced with alanines, I showed that Pcp1 CM1 region is required for its function. Moreover, using CM1 regions from two human proteins that are implicated in schizophrenia and microcephaly development, MMGL and CDK5RAP2, I showed that human CM1 domains could rescue yeast protein function, demonstrating that the CM1 region is conserved across evolution. In the second part of my project, I focused on regulation of cytoplasmic MT nucleation. In fission yeast, cytoplasmic MT nucleation occurs from several distinct sites in the cell and is promoted by the Mto1/2 complex. The Mto1/2 complex is composed of multiple copies of Mto1 and Mto2 and interacts with the γ-TuC. Disruption of the interaction of Mto1/2 with the γ-TuC, or of the Mto1-Mto2 interaction, results in a complete loss of interphase cytoplasmic nucleation. As cells enter mitosis, Mto2 is hyperphosphorylated, and the Mto1-Mto2 interaction is disrupted, leading to abolishment of cytoplasmic nucleation. This led to a hypothesis that Mto2 phosphorylation regulated the Mto1/2 complex mitotic disassembly. I showed that Mto2 phosphorylation is used to control levels of cytoplasmic nucleation in both interphase and mitosis. During interphase, I found that Mto2 is phosphorylated in order to reduce levels of MT nucleation. When Mto2 phosphorylation is prevented by mutation of phosphorylatable residues to alanines, Mto1/2 mutant complexes show a more robust interaction with the γ-TuC, and more MTs are nucleated in the cytoplasm. During mitosis, hyperphosphorylation of Mto2 plays a role in the disassembly of Mto1/2 complexes. In particular, while the interaction of wild-type Mto2 with Mto1 is disrupted during mitosis, Mto2-alanine mutants, in which phosphorylation was nearly abolished, are still able to interact with Mto1 in mitosis. Interestingly, Mto1/2 complexes containing Mto2-alanine mutants are still disassembled in mitosis by disruption of Mto2 self-interaction. I used SILAC phosphoproteomics to show that Mto2-alanine is still phosphorylated in mitosis, suggesting the Mto2 self-interaction might also be controlled by phosphorylation. While doing so, I developed a novel SILAC quantification method that is particularly useful for quantification of multiply phosphorylated proteins and peptides. Using data obtained by SILAC, I generated additional Mto2 alanine mutants with more phosphorylation sites mutated. Preliminary analysis showed that these mutants are similar to the alanine mutants analysed previously; however, more analysis is required to generate more definitive conclusions. In summary, in this study I have uncovered the functional conservation of the CM1 region from yeast to human. I also showed that Mto2 phosphorylation regulates cytoplasmic MT nucleation in both interphase and mitosis, by regulating the Mto2-Mto1 interaction and the Mto2-Mto2 self-interaction and therefore remodelling the Mto1/2 complex.

As quinases dependentes de ciclinas são proteínas que podem ser divididas de acordo com a sua atuação no ciclo celular ou no controle transcricional, elas se tornam ativas em determinadas etapas do ciclo celular dependendo do seu grau de fosforilação e de sua ligação com ciclinas e proteínas inibitórias, e exercem sua função fosforilando outras proteínas envolvidas no ciclo de divisão celular e transcrição influenciando suas atividades, garantindo que cada processo do ciclo ocorra em uma sequência ordenada. A CDK13 faz parte da família de proteínas quinases dependentes de ciclina, pode se ligar a ciclinas do tipo L ou K, regula os eventos de \"splicing\" alternativo, e interage com a proteína Tat do vírus HIV atuando como um possível fator de restrição, sendo que sua superexpressão diminui a produção de algumas proteínas virais suprimindo a produção do vírus. O DNA referente à CDK13 é replicado em células cancerosas, principalmente dos tipos hepático e cólon e reto, sendo um alvo para inibidores para tratamento de câncer. A fim de contribuir para o estudo dessa proteína, o projeto tem como objetivo expressá-la utilizando métodos de tecnologia de DNA recombinante. A sequência de DNA referente à CDK13 foi amplificada pela reação em cadeia da polimerase, após sua purificação, foi inserida no vetor pCR-Blunt e clonada em células de E. coli DH5α competentes. Porém, o DNA não foi liberado pela reação com as enzimas de restrição BamHI e NdeI. As bactérias Rosetta(DE3) transformadas com um plasmídeo sintético e crescidas em meio de auto-indução expressaram a CDK13. Após lise celular e purificação em coluna de Ni2+, a proteína foi detectada por Western Blot. Já as bactérias Rosetta(DE3) transformadas com o plasmídeo sintético modificado (o qual compreende a região do DNA que expressa o bolsão de ligação da CDK13), e induzidas em meio LB expressaram a CDK13, porém não foi possível purificá-la em coluna de afinidade ao Ni2+. / The cyclin-dependent kinases are proteins that can be classified by their function in the cell cycle or transcriptional control. They are activated in particular steps of the cell cycle depending on their phosphorylation degree, cyclin binding and inhibitory proteins. They act phosphorylating other proteins involved in the cell cycle and transcriptional control, influencing in their activities, ensuring that each step of the cell cycle occur in an ordered sequence. The CDK13 is one of the cyclin-dependent kinases family member, it can bind to L or K cyclins, regulates the alternative splicing and interact with HIV Tat protein, acting as a possible restriction factor, its overexpression decreases the production of some viral proteins, and suppresses the virus production. The DNA corresponding to CDK13 is replicated in cancer cells, mainly of hepatic and colon rectal types; therefore it is a target for inhibitors for cancer therapy. In order to contribute for the studies of this protein, the goal of the project is to express it using methods of recombinant DNA technology. The DNA sequence corresponding to CDK13 was amplified by polymerase chain reaction, after its purification, it was inserted to pCR-Blunt vector and cloned into E. coli DH5α competent cells. However, the DNA wasn\'t released by the BamHI and NdeI restriction enzymes. The Rosetta(DE3) cells transformed with a synthetic plasmid pET28a::CDK13 and grown in auto-induction media expressed the CDK13. After cell lysis and purification by Ni2+ affinity colum, the protein was identified by Western Blot. However, the Rosetta(DE3) cells transformed with the modified synthetic plasmid (that comprehends the DNA region which expresses the binding pocket region) induced in LB media, expressed the CDK13. Yet, it wasn\'t possible to purify the protein in the Ni2+ affinity column.

CD40 is a TNF-Receptor (TNFR) superfamily member that functions to promote several facets of the humoral immune response--including B cell proliferation, differentiation, antibody isotype switching, and cytokine expression. TNFR superfamily members lack intrinsic kinase activity and must recruit members of the TNFR-associated factor (TRAF) family of adaptor proteins to connect the receptor to intracellular signaling pathways. CD40-mediated JNK and NF-κB activation is critical for an intact humoral immune response; however, the precise mechanisms governing the spatiotemporal activation of these pathways are not completely understood. In this study we report that CD40 ligation results in the dual phosphorylation of TRAF2 on Ser-11 and Ser-55 to control the subcellular localization of key pathway intermediates and temporally regulate downstream JNK and IKK/NF-κB pathway activation. Notably, TBK1- mediated TRAF2 Ser-11 phosphorylation elicits the dissociation of a signaling complex, consisting of TRAF2, cIAP1/2, and IKKγ, from the CD40 receptor to potentiate a secondary phase of JNK and IKK activation. In the absence of this phosphorylation event, these proteins translocate to the insoluble lipid rafts along with the membrane-bound receptor complex, where TRAF2 undergoes Ser-55 phosphorylation-dependent self-ubiquitination and degradation necessary for cessation of JNK activation. Furthermore, TRAF2 Ser-11 phosphorylation inhibits non-canonical NF-κB activation by promoting the lipid raft localization of the CD40 receptor complex. This suggests that TRAF2 dual phosphorylation acts as a molecular switch to control canonical and non-canonical NF-κB activation. CD40 signaling is heavily implicated in a wide array of chronic inflammatory and autoimmune diseases--including Alzheimer's, Grave's disease, and diabetes. As such, characterization of the molecular mechanisms directing CD40 signal transduction will provide a foundation for the further development of targeted immunomodulatory therapeutics. In addition, the NF-κB transcriptional program has well-defined roles in oncogenesis and tumor progression, and many B cell lymphomas exploit the CD40L/CD40 dyad to constitutively activate the NF-κB pathway and potentiate neoplastic growth and survival. Through these analyses, we demonstrate that TRAF2 phosphorylation on Ser-11 and Ser-55 promote cell survival in response to genotoxic and oxidative stress, respectively, by regulating JNK and NF-κB pathway activation and coordinating the subcellular localization and stability of key signaling effectors. Furthermore, we show that inhibition of TRAF2 phosphorylation in B-cell lymphoma cells increases their sensitivity to standard frontline chemotherapeutics, including doxorubicin and vincristine, as well as the novel agents bortezomib and arsenic trioxide. These findings are clinically significant, as TRAF2 is found over-expressed and constitutively phosphorylated in DLBCL cell lines and patient biopsies. In addition, mice bearing tumors that harbor TRAF2 Ser-11 phospho-null mutations are more responsive to treatment with doxorubicin and have significantly prolonged survival compared to wild-type counterparts in a syngeneic model of B-cell lymphoma. The tumor microenvironment is characterized by pro-inflammatory cytokines, hypoxia, low glucose, and free radicals, all of which are known to induce chronic cellular stress and NF-κB activation. Cancer cell adaptation to these stressors has profound consequences for malignant progression and therapeutic response. In this regard, our findings present a unique opportunity where the molecular targeting to TRAF2 phosphorylation could increase the efficacy of current therapies by suppressing basal NF-κB activation, thus synergistically sensitizing NF-κB-driven malignancies to chemotherapeutic-induced cell death.

The lens is responsible for focusing light into the retina. It accomplishes this through its maturation from an epithelial cell into a fiber cell. A large amount of research has been done on cellular differentiation. Nevertheless, we still lack knowledge on many different aspects of differentiation, including a complete theory on the mechanism behind differentiation. Due to the lens’ unique structure and cell types, this is an ideal model for studying differentiation. Our research has shown that αB crystallin, a small heat shock protein, is able to modulate cytochrome C levels and protect the mitochondria under oxidative stress. Also, cytochrome C release is often followed by caspase 3 activation. In addition, research has shown that low levels of caspase 3 activation is essential in driving differentiation. My work examined if αB crystallin could modulate cytochrome C to lower caspase 3 levels to allow for differentiation rather than apoptosis. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Adherens junctions are essential for embryogenesis and tissue homeostasis. The major transmembrane adhesion receptors in adherens junctions are the cadherins, which mediate cell-cell adhesion by binding to cadherins on adjacent cells. Cadherin function is regulated by the protein complexes that assemble at its cytoplasmic tail. Vinculin is one cytoplasmic component of the cadherin adhesion complex, but unlike other junction components, it also is enriched in cell-matrix adhesions. The presence of vinculin in cellmatrix adhesions has commanded the most attention, while little is known about its role in cell-cell adhesions. To define the role of vinculin in adherens junctions, I established a short hairpin RNA-based knockdown/substitution system that perturbs vinculin preferentially at sites of cell-cell adhesion. When this system was applied to epithelial cells, cell morphology was altered, and cell-cell adhesion was reduced owing to a lack of cadherin on the cell surface. I investigated the mechanism for this effect and found that vinculin must bind to beta-catenin to regulate E-cadherin surface expression. Having established a role for vinculin in cell-cell adhesions, the critical question became how vinculin recruitment to and activation at cell-cell junctions are regulated. I found that á-catenin triggers activating vinculin conformational changes. Unlike all of the known vinculin activators in cell-matrix adhesions, alpha-catenin binds and activates vinculin independently of an A50I substitution. Thus, adherens junction activators and cell-matrix activators bind to distinct regions of vinculin to activate this molecule. Using mutant vinculins that cannot be tyrosine phosphorylated, I found that vinculin recruitment to cell-cell adhesions, but not cell-matrix adhesions, requires phosphorylation at Y822. Furthermore, this residue is phosphorylated by Abl tyrosine kinases during the assembly of cell-cell adhesions. Taken together, these studies explain how vinculin is differentially recruited to adherens junctions and cell-matrix adhesions and describes the first known role for vinculin at cell-cell adhesions.

Mitochondria are important cellular organelles whose functions include generation of ATP, sequestration and release of pro-apoptotic molecules and calcium buffering. Mitochondria function is tightly linked to organelle morphology, which exits in a dynamic spectrum between a highly interconnected/fused mitochondria network to a punctate/fragmented scattering of individual mitochondrion. A family of large GTPase enzymes modulates this spectrum, with fusion catalyzed through the actions of mitofusin 1 and 2 (Mfn1/2) on the outer mitochondria membrane (OMM) and optic atrophy 1 (Opa1) causing fusion of the inner mitochondria membrane (IMM). On the other end of the spectrum, fragmentation is catalyzed through the actions of dynamin-related protein 1 (Drp1). Drp1 is recruited from the cytosol to binding partners at the OMM, organizes into concentric spiral rings, undergoes GTP hydrolysis to constrict the ring and pinches mitochondrion in two. While fragmentation is achieved through the action of only one GTPase enzyme, the mechanisms behind the complex regulation of Drp1 remain relatively obscure. In order to expand upon known Drp1 regulatory mechanisms, an examination of how both Drp1 splicing and Drp1 recruitment to the OMM contributes to protein regulation is necessary. Drp1 contains three alternatively spliced exons, resulting in the potential generation of eight protein isoforms. Each of these isoforms is capable of inducing mitochondrial fragmentation, however one exon arrangement (termed Drp1-x01) can also bind to microtubules within the cell. Characterization of the Drp1-x01 isoform at both the RNA and protein level indicate an important, yet incompletely characterized, role in immune system biology. Drp1 is capable of interacting with several proteins localized at the OMM. Among these, mitochondria fission factor (Mff) has been implicated in the formation of Drp1 spirals and the eventual fragmentation process. Mff contains four alternatively spliced exons as well as several phosphorylation sites identified through nonbiased phosphoproteomic screens. Inclusion of alternative exons to the Mff structure decreases its ability to recruit Drp1 from the cytosol, while phosphomimetic substitutions to conserved serine residues enhances the Drp1::Mff interaction. Taken together, this suggests that regulation of mitochondrial fragmentation occurs at the pretranslational (alternative splicing) and the posttranslational (phosphorylation) level is critical for maintaining the complex, yet essential, balance between mitochondrial fission and fragmentation.

The purpose of this thesis was to investigate mechanisms contolling the growth of corneal epithelial cells, particularly the intracellular signals involved with stratification compared with cellular migration and maturation. Buttons of epithelium were cultured in different culture media. The explants were monitored microscopically for their growth patterns and finally fixed and examined for cytokeratin, vimentin and actin. Different growth patterns were observed in the different media, indicating that different signalling patterns must be operating in these cells depending upon the media in which they were grown. To investigate the intracellular pathways controlling the different growth patterns, the protein phosphorylation of different cultures was investigated. The two proteins, p57 and p30, are strongly suggested to be associated with stratification of the epithelial cells. The possible involvement of the common serine kinase, PKC, in controlling the growth pattern of corneal epithelial cells were also investigated. The results suggested that an intracellular pathway involving PKC promotes the maturation and spread of the cells but is not involved in their stratification. These experiments taken together indicate that the different aspects of corneal epithelia cell growth are tightly controlled and may occur quite independently. Specific protein expression appears to be important for stratification, and phosphorylation of proteins by PKC appears to be involved with the maturation of epithelial cells from basal cells. It also indicates that the mature cells are capable of producing the extracellular matrix protein fibronectin which appears to have an important role in causing the spread as distinct from the stratification of the corneal epithelial cells. / Doctor of Philosophy (Ph.D.)

We have developed a novel framework that can be applied for the analysis of signal transduction networks, both to facilitate reconstruction of the network structure and quantitatively characterize the interaction between network components. This approach, termed activation ratio analysis, involves the ratio between active and inactive forms of signaling intermediates at steady state. The activation ratio of an intermediate is shown to depend linearly upon the concentration of the activating enzyme. The slope of the line is defined as the activation factor, and is determined by the kinetic parameters of activation and inactivation. When activation ratios for simple signaling systems are considered, a set of rules develop that can be used to transform a set of experimental data to a proposed model network structure, with activation factors yielding a measure of activation potential between intermediates. / Singapore-MIT Alliance (SMA)

Thesis (M.S. in Cancer Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 160-193).