ABSTRACT The Golgi apparatus has always been an interesting organelle of study because of its unique morphology as well as the critical roles it plays in cell biology. It is situated next to the endoplasmic reticulum and secreted proteins must pass through the Golgi vesicular pathway for modifications and targeting. In addition, the Golgi apparatus plays an essential role in establishing cellular polarity. Cell polarity refers to difference in orientation of cell structures spatially, and is involved in establishing functionality. The Golgi apparatus establishes cell polarity in various ways including orienting itself spatially, biasing vesicular trafficking within the cell, and most importantly through its role as a microtubule organizing center. The cytoskeleton provides the structural framework for cells. Microtubules nucleated from the Golgi-dependent microtubule organizing center result in an asymmetric cytoskeleton. An asymmetric cytoskeleton is essential to establishing cell polarity. Neurons require cell polarity to establish the essential structures such as the axon and dendrites. The Golgi apparatus establishes neuronal polarity through its extensive network of associated proteins. In this review, we will discuss the growing evidence supporting the role of the Golgi apparatus in establishing neuronal polarity.

Epithelial cells are unique for their ability to strongly adhere to one another and coordinate communication across an asymmetrical, polar plasma membrane. These properties are necessary for carrying out normal epithelial function, such as absorbing/secreting molecules, repairing wounds, lining organs, etc. Cadherins, claudins, and occludins are major players of epithelial cell adhesion and polarity. Previously, transmembrane immunoglobulin domain containing-1, TMIGD1, was identified as a novel cell adhesion molecule, whose expression is downregulated in human renal carcinomas. Re-expression of TMIGD1 in renal tumor cells resulted in altered cell morphology and inhibition of tumor growth. In this study, we examined the hypothesis that TMIGD1 activity is associated with epithelial cell polarity. We demonstrated that TMIGD1 regulates actin stress fibril formation. A 3-dimensional (3D) cell culture assay was developed to examine the role of TMIGD1 in cell morphology and polarity. Our results demonstrate that TMIGD1 regulates actin fibril formation in Madin-Darby Canine Kidney (MDCK) cells, as blocking TMIGD1 activity by blocking antibody inhibited actin fibril formation in 3D cell culture system. Moreover, ectopic expression of TMIGD1 in rectal carcinoma cells, (RKO) , significantly inhibited filopodia formation. Taken together, our data identifies TMIGD1 as a possible regulator of epithelial cell morphology and polarity. / 2020-07-03T00:00:00Z

Animal cells, as distinct as epithelia and migratory cells, have cell polarity that is defined by a common set of molecules. The Par complex polarizes the cortex of animal cells through the activity of atypical protein kinase C (aPKC). In this work, I aimed to determine the mechanism of aPKC substrate polarization and identify common characteristics of aPKC substrates that are polarized by phosphorylation. I found that several diverse Par-polarized proteins contain short highly basic and hydrophobic motifs that overlap with their aPKC phosphorylation sites. These Phospho-Regulated Basic and Hydrophobic (PRBH) motifs mediate plasma membrane localization by electrostatics-based phospholipid binding when unphosphorylated but are displaced into the cytoplasm when phosphorylated. To assess whether the Par complex polarizes other proteins by this mechanism, I developed an algorithm to identify potential PRBH motifs and score these linear motifs for basic and hydrophobic character, as well as the quality and number of aPKC phosphorylation sites. Using this algorithm, I identified numerous putative PRBH candidates in the fruit fly proteome and performed two screens of these candidates for Par-polarized proteins. The first screen focused on determining whether aPKC regulates cortical targeting of proteins that are reported to be polarized. This screen identified the Rho GAP crossveinless-c (cv-c) to be a novel aPKC substrate and found that aPKC is sufficient to polarize cv-c in a reconstituted polarity assay. The second screen characterized the localization of putative PRBH motif-containing proteins in vivo. This screen identified a previously uncharacterized protein, CG6454, to be basolateral in epithelia; however, ex vivo experiments found it to have a Ca2+-dependent and aPKC-independent membrane targeting mechanism. Overall this work identified a common mechanism for Par substrate polarization and used knowledge of this mechanism to identify a novel Par effector. This dissertation contains previously published coauthored materials as well as unpublished materials. / 2019-05-08

Two groups of growth regulators were described in Drosophila imaginal disc cell culture Cl.8+. Imaginal disc growth factors (IDGFs) belonging to chitinase-like protein family of carbohydrate binding proteins and Adenosine deaminase-related growth factors (ADGFs), which are active adenosine deaminases influencing homeostasis of key cellular metabolite adenosine. The functions of two of the IDGFs, as well as the effects of extracellular adenosine and its receptor were studied primarily in in vitro cell culture. Our results supported their roles in the regulation of cell survival and energy homeostasis especially in imaginal disc cells. Both the IDGFs and adenosine also play important roles in organismal responses to stress and infection and may interact in vivo.

xiii, 48 p. : (col. ill.) A print copy of this title is available through the UO Libraries under the call number: SCIENCE QL537.D76 N57 2007 / Asymmetric cell division (ACD) is a unique mechanism employed during development to achieve cellular diversity from a small number of progenitor cells. Cells undergoing ACD distribute factors for self-renewal at the apical cortex and factors for differentiation at the basal cortex. It is critical for proper development that the mitotic spindle be tightly coupled to this axis of polarization such that both sets of proteins are exclusively segregated into the daughter cells. We use ACD in Drosophila neuroblasts as a model system for understanding the molecular mechanisms that govern spindle-cortical coupling. Neuroblasts polarize Partner of Inscuteable (Pins), Gαi and Mushroom Body Defect (Mud) at the apical cell cortex during mitosis. Gαi and Pins are required for establishing cortical polarity while Mud is essential for spindle-cortical alignment. Gαi and Mud interact through Pins GoLoco domains and tetratricopeptide repeats (TPR) respectively, however it is unclear how Mud activity is integrated with Pins and Gαi to link neuroblast cortical polarity to the mitotic spindle. This dissertation describes how Pins interactions with Gαi and Mud regulate Iwo fundamental aspects of neuroblast ACD: cortical polarity and alignment of the spindle with the resulting polarity axis. I demonstrate that Pins is a dynamic scaffolding protein that undergoes a GoLoco-TPR intramolecular interaction, resulting in a conformation of Pins with low Mud and reduced Gαi binding affinity. However, Pins TPR domains fail to completely repress Gαi binding, as a single GoLoco is unaffected by the intramolecular isomerization. Gαi present at the apical cortex specifies Pins localization through binding this "unregulated" GoLoco. Liberation of Pins intramolecularly coupled state occurs through cooperative binding of Gαi and Mud to the other GoLoco and TPR domains, creating a high-affinity Gαi-Pins-Mud complex. This autoregulatory mechanism spatially confines the Pins-Mud interaction to the apical cortex and facilitates proper apical-spindle orientation. In conclusion, these results suggest Gαi induces multiple Pins states to both properly localize Pins and ensure tight coupling between apical polarity and mitotic spindle alignment. / Adviser: Ken Prehoda

xi, 93 p. ; ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / The atypical protein kinase C (aPKC) protein has been implicated in several human tumors yet very little is known about how aPKC is regulated. One mechanism that has been proposed as the possible source of several types of tumor is the defective asymmetric cell division of a small number of tumor stem cells. aPKC is required for cell polarization from nematodes to mammals, in tissues as diverse as epithelia, embryonic blastomeres, and neural progenitors. In Drosophila central nervous system, mitotic neural stem cells, termed neuroblasts, recruit the polarity proteins aPKC at the cell apical cortex. pack restricts the localization of the differentiation factors Miranda, Prospero, Brat, and Numb to the cell's basal cortex. Later during mitosis, the cytokinetic furrow sets unevenly about the neuroblast apical-basal axis to produce a large cell (neuroblast) which will continue to divide and self-renew, while the smaller ganglion mother cell inherits differentiation factors and terminally divides to give rise to a pair of neurons and/or glia. Asymmetric cell division is not only critical for generating cellular diversity, it also ensures that a stable population of neural stem cell is constantly maintained while allowing neurogenesis to occur. Despite its conserved role in cell polarity and tumorigenesis, relatively little is known about aPKC regulators and targets. In a co-authored work, I show that the small Rho GTPase, Cdc42, indirectly regulates aPKC. However, this stimulation is modest and the mutant phenotypes are not fully penetrant suggesting that other regulators exist. To isolate other aPKC regulators and targets, I used a biochemical approach to identify aPKC-interacting proteins, and identified one positive regulator and one negative regulator of aPKC. I show that Dynamin-associated protein-160 (Dap160; related to mammalian Intersectin) is a positive regulator of aPKC. I also show that a regulatory subunit of protein phosphatase 2A (PP2A), negatively regulates aPKC. This dissertation includes both my previously published and my co-authored material. / Adviser: Chris Doe

Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle. I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh). I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent. This dissertation includes unpublished and previously published co-authored material. / 10000-01-01

O problema de fatorar e decompor funções Booleanas é Σ-completo2 para funções gerais. Algoritmos eficientes e exatos podem ser criados para classes de funções existentes como funções read-once, disjoint-support decomposable e read-polarity-once. Uma forma fatorada é chamada de read-once (RO) se cada variável aparece uma única vez. Uma função Booleana é RO se existe uma forma fatorada RO que a representa. Por exemplo, a função representada por =12+134+135 é uma função RO, pois pode ser fatorada em =1(2+3(4+5)). Uma função Booleana f(X) pode ser decomposta usando funções mais simples g e h de forma que ()=ℎ((1),2) sendo X1, X2 ≠ ∅, e X1 ∪ X2 = X. Uma decomposição disjunta de suporte (disjoint-support decomposition – DSD) é um caso especial de decomposição funcional, onde o conjunto de entradas X1 e X2 não compartilham elementos, i.e., X1 ∩ X2 = ∅. Por exemplo, a função =12̅̅̅3+123̅̅̅ 4̅̅̅+12̅̅̅4 é DSD, pois existe uma decomposição tal que =1(2⊕(3+4)). Uma forma read-polarity-once (RPO) é uma forma fatorada onde cada polaridade (positiva ou negativa) de uma variável aparece no máximo uma vez. Uma função Booleana é RPO se existe uma forma fatorada RPO que a representa. Por exemplo, a função =1̅̅̅24+13+23 é RPO, pois pode ser fatorada em =(1̅̅̅4+3)(1+2). Esta tese apresenta quarto novos algoritmos para síntese de funções Booleanas. A primeira contribuição é um método de síntese para funções read-once baseado em uma estratégia de divisão-e-conquista. A segunda contribuição é um algoritmo top-down para síntese de funções DSD baseado em soma-de-produtos, produto-de-somas e soma-exclusiva-de-produtos. A terceira contribuição é um método bottom-up para síntese de funções DSD baseado em diferença Booleana e cofatores. A última contribuição é um novo método para síntese de funções RPO que é baseado na análise de transições positivas e negativas. / The problem of factoring and decomposing Boolean functions is Σ-complete2 for general functions. Efficient and exact algorithms can be created for an existing class of functions known as read-once, disjoint-support decomposable and read-polarity-once functions. A factored form is called read-once (RO) if each variable appears only once. A Boolean function is RO if it can be represented by an RO form. For example, the function represented by =12+134+135 is a RO function, since it can be factored into =1(2+3(4+5)). A Boolean function f(X) can be decomposed using simpler subfunctions g and h, such that ()=ℎ((1),2) being X1, X2 ≠ ∅, and X1 ∪ X2 = X. A disjoint-support decomposition (DSD) is a special case of functional decomposition, where the input sets X1 and X2 do not share any element, i.e., X1 ∩ X2 = ∅. Roughly speaking, DSD functions can be represented by a read-once expression where the exclusive-or operator (⊕) can also be used as base operation. For example, =1(2⊕(4+5)). A read-polarity-once (RPO) form is a factored form where each polarity (positive or negative) of a variable appears at most once. A Boolean function is RPO if it can be represented by an RPO factored form. For example the function =1̅̅̅24+13+23 is RPO, since it can factored into =(1̅̅̅4+3)(1+2). This dissertation presents four new algorithms for synthesis of Boolean functions. The first contribution is a synthesis method for read-once functions based on a divide-and-conquer strategy. The second and third contributions are two algorithms for synthesis of DSD functions: a top-down approach that checks if there is an OR, AND or XOR decomposition based on sum-of-products, product-of-sums and exclusive-sum-of-products inputs, respectively; and a method that runs in a bottom-up fashion and is based on Boolean difference and cofactor analysis. The last contribution is a new method to synthesize RPO functions which is based on the analysis of positive and negative transition sets. Results show the efficacy and efficiency of the four proposed methods.

This study explores a category of expressions akin to not yet, already, still and no longer, called Phasal Polarity (PhP) expressions. They encode the domains of phasal values, polarity and speaker expectations and have previously been described in European languages (van der Auwera 1998) and in a small, genealogically diverse sample (van Baar 1997). Using reference grammars as the primary source of information, the aim of this crosslinguistic study is to describe PhP expressions in a sample of East Bantu languages. It is found that the distribution and behaviour of PhP expressions in East Bantu differ from both European languages and the genetically diverse sample of van Baar. The markers are found to be morphologically diverse and of varied crosslinguistic frequency. Furthermore, the verbal morphotax indicates that the markers are, or are in the process of, being incorporated into the tense-aspect systems of their respective language. / Denna studie utforskar en kategori av utryck besläktade med inte än, redan, fortfarande och inte längre, som kallas Phasal Polarity (PhP) expressions. De uttrycker fas, polaritet och talar förväntningar och har tidigare beskrivits i en studie av europeiska språk (van der Auwera 1998) och i en småskalig, genetiskt mångfaldig studie (van Baar 1997). Med referensgrammatikor som primärkälla ämnar denna studie undersöka PhP expressions i ett urval av språk i underfamiljen Östbantu. Resultaten visar att PhP expressions i Östbantu har annorlunda distribution och användning jämfört med europeiska språk och van Baars urval. Markörerna varierar avseende morfologi och tvärspråklig frekvens. Verbens morfotax indikerar att markörerna har, eller håller på att, inkorporeras i respektive språks system.

Como intuito de contribuir para a elucidação do mecanismo de quimi-excitação da reação peroxi-oxalato, a transformação quimiluminescente mais eficiente conhecida que envolve a versão intermolecular do mecanismo \"Chemically Initiated Electron Exchange Luminescence\" - CIEEL, foram efetuados estudos cinéticos e determinados os rendimentos quânticos de quimi-excitação desta reação em diferentes solventes e misturas de solventes. A cinética da reação de oxalato de bis-2,4,6-triclorofenila com peróxido de hidrogênio, catalisada por imidazol foi estudada na presença de 9,10-difenilantraceno como ativador (ACT) em diferentes solventes. O comportamento das constantes de velocidade observados em função da polaridade e viscosidade dos solventes indica que o mecanismo dos passos iniciais desta transformação até a formação do intermediário de alta energia (IAE) não sofre modificações drásticas em função das propriedades de solventes, permitindo a escolha de solventes e misturas de solventes adequados para o estudo do mecanismo de quimi-excitação do sistema. A influência da viscosidade sobre os rendimentos quânticos singlete (Φs) e os rendimentos quânticos singlete na concentração infinita do ACT (Φs∞ - obtidos de gráficos duplo recíprocos entre os (Φs) e a [ACT]) foi estudada em misturas entre acetato de etila e ftalato dimetilico (que possuem viscosidades diferentes e parâmetros de polaridade similares, além de levarem a rendimentos quânticos altos), indicando um aumento considerável dos rendimentos quânticos singlete com o aumento da viscosidade do meio. Os dados foram tratados com base nos modelos colisional e friccional, obtendo indicação clara da operação de um efeito de gaiola do solvente no processo de quimi-excitação da reação neste sistema de alta eficiência. A influência da polaridade do meio sobre os parâmetros de quimi-excitação da reação peroxi-oxalato foi analisada, de maneira análoga, em misturas entre tolueno e acetato de etila e entre acetato etila e acetona. Os rendimentos quânticos singlete aumentam inicialmente em função da polaridade do meio até um valor máximo, a partir do qual eles sofrem diminuição com o aumento da polaridade. Este comportamento indica a melhor solvatação dos pares de íons radicais pelo aumento da polaridade de solvente levando ao aumento dos rendimentos, entretanto, polaridades maiores do meio podem levar à formação de pares de íons separados por solvente e íons separados, desta maneira diminuindo a eficiência da quimi-excitação. Os parâmetros de quimiluminescência da reação peroxi-oxalato foram ainda estudados em uma série de solventes puros com diferentes valores de polaridade e viscosidade. Os rendimentos quânticos obtidos foram correlacionados com os parâmetros de viscosidade, polaridade e polarizabilidade, utilizando-se uma regressão linear múltipla. Deste ajuste se pode confirmar que a viscosidade exerce a maior influência sobre os rendimentos quânticos e o aumento da polaridade contribui também para maiores rendimentos, entretanto, os parâmetros de ajuste contêm erros muito grandes devido à dispersão dos valores. Contrariamente, o aumento da polarizabilidade do meio parece diminuir a eficiência de quimi-excitação. / In order to contribute to the elucidation of the chemiexcitation mechanism of peroxyoxalate reaction, the most efficient chemiluminescence reaction known that involves the intermolecular \"Chemically Initiated Electron Exchange Luminescence\" - CIEEL mechanism, kinetically studies were performed and chemiexcitation quantum yields determined in different solvents and solvent mixtures. The reaction kinetics of bis(2,4,6-trichlorophenyl) oxalate with hydrogen peroxide catalyzed by imidazole in the presence of 9,10-diphenylanthracene as an activator (ACT) was studied in different solvents. The behavior of the observed rate constants in function of solvents polarity and viscosity indicates that the initials reaction steps leading to high energy intermediate formation (HEI) do not change drastically in function of the solvent properties, allowing to choose suitable solvents and solvent mixtures for a study of its the chemiexcitation mechanism. The viscosity influence on the singlet quantum yields (Φs) and singlet quantum yields at infinite ACT concentration (Φs∞ - obtained from double reciprocal plots between Φs and [ACT]) was studied in mixtures of ethyl acetate and dimethyl phthalate (which have different viscosities and similar polarity parameters), indicating a considerable increasing in singlet quantum yields with the increasing of medium viscosity. Data were analyzed by the collisional and the free volume model, which clearly indicates the occurrence of a solvent cage effect in the chemiexcitation process of this high efficient system. The influence of medium polarity on the chemiexcitation parameters of the peroxyoxalate reaction were studied, in a similar way, in mixtures of toluene and ethyl acetate and in mixtures of ethyl acetate and acetone. Singlet quantum yields increase in function of medium polarity for low polar solvents, reaching a maximum value, where a further polarity increase leads to a decrease in quantum yields. This behavior indicates better solvation of radical íon pairs by solvent polarity increase, leading to increased quantum yields, however, higher solvent polarities can lead to the formation of solvent separated ion pairs and individually solvate ions, thereby decreasing chemi-excitation efficiency. Chemiluminescence parameters of the peroxyoxalate reaction were also studied in pure solvents with different viscosities and polarities. The obtained singlet quantum yields were correlated with viscosity, polarity and polarizability parameters by multiple linear regression analysis. The correlations obtained show that viscosity exerts the most significant influence on the quantum yields and an increase in solvent polarity also contributes to higher quantum yields; however, these fitting parameters possess high standard deviations due to the considerable dispersion of quantum yield values. Contrary, an increase in medium polarizability appears to decrease the chemi-excitation efficiency.