Life Sciences Commons^™

Investigation Of Shot1-Binding Atpases In Arabidopsis Thaliana, Sam Zelman

Masters Theses

Mitochondria play critical roles not only in primary metabolism as a central organelle for ATP generation, but also in responding to abiotic stresses. We identified a mutation in the MTERF18 (Mitochondrial Transcription Termination factor)/SHOT1 (Suppressor of hot1-4 1) gene in Arabidopsis thaliana that enables plants to better tolerate heat and oxidative stresses, presumably due to reduced oxidative damage, but the exact molecular mechanism of the heat tolerance is unknown. In order to reveal the stress tolerance mechanisms of mterf18/shot1 mutations, it is critical to understand the molecular defects of the mutant and to identify …

Exploration Of The Association Between Muscle Volume And Bone Geometry Reveals Surprising Relationship At The Genetic Level, Prakrit Subba

Masters Theses

The evolution of jaws in cichlid fishes of the East African Great Lakes is a textbook example of adaptive radiation in vertebrates. Karl Liem postulated that this adaptive radiation has been possible due to the functional decoupling of two cichlid functional units – the pharyngeal jaw (PJ) and the oral jaw (OJ). This functional decoupling of the jaws has enabled the OJ to be relieved of its dual role of prey capturing and processing and has allowed the PJ to take on the role of prey processing. As a result, African cichlids have adapted the morphology of their functional units …

Assessing Stress Tolerance Of Organelle Small Heat Shock Protein Mutants In Arabidopsis Thaliana, Parth Patel

Masters Theses

Molecular chaperones are proteins found in virtually every organism and are essential to cell survival. When plants are heat stressed, they upregulate and downregulate multiple genes, many of which are associated with the heat shock response. Small heat shock proteins (sHSPs) are one class of molecular chaperones that are upregulated during heat shock. They are proposed to act as the first line of defense by binding to heat sensitive proteins and preventing their irreversible aggregation. However, many details of sHSP function remain to be discovered and exactly what proteins they protect is unresolved. In addition to cytosolic sHSPs found in …

The Shape Of U: Mapping Out Protective Elements In Mrna Escapees, Jacob Miles

Masters Theses

A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the …

Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois

Doctoral Dissertations

Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …

Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther

Doctoral Dissertations

Many legumes form a symbiosis with nitrogen-fixing bacteria found in the soil. This relationship is beneficial to both the plant and the bacteria; the plant receives nitrogen that is otherwise limited, and the bacteria receive fixed carbon. Upon sensing the bacteria, the plant forms a new organ (the nodule) where the bacteria are housed within the cells. Many genes are required for the proper formation and function of nodules; this dissertation is broadly focused on how genes required for nitrogen-fixing symbiosis are co-opted from other cellular processes and how they are specialized for symbiosis. Protein trafficking from the plant to …

Structure Function Analysis Of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles In Kinetoplast Maintenance, Jonathan Miller

Doctoral Dissertations

Kinetoplastid organisms including medically relevant species like Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy number and sequence classes among different species, kDNA is most often found as a network of thousands of DNA molecules catenated together. This unique biological property of disease-causing organisms has been a subject of study as a potential drug target since it is essential for parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and related diseases in other mammals and …

Involvement Of Calcium-Dependent Protein Kinases And Phosphatases In Sperm Capacitation-Associated Events, Bidur Paudel

Doctoral Dissertations

ABSTRACT To acquire fertilizing ability, mammalian sperm undergo a series of biochemical and physiological changes collectively known as capacitation^1,2. At the molecular level, capacitation is associated with a fast bicarbonate (HCO₃^-)-dependent activation of a unique type of soluble adenyl cyclase (sAC) and a consequent increase in cyclic AMP (cAMP) levels and PKA activation³. Activation of a cAMP/PKA pathway results in the phosphorylation of PKA substrates, which in turn initiates activation of several signaling cascades ultimately leading to an increase in phosphorylation on tyrosine residues (P-Tyr) of sperm axonemal proteins^4,5. Increase in …

Gene Expression Regulation In The Mouse Liver By Mechanistic Target Of Rapamycin Complexes I And Ii, Anthony Poluyanoff

Masters Theses

The mechanistic target of rapamycin (mTOR) is a key serine/threonine protein kinase that functions in complexes mTORC1 and mTORC2. mTORC1, originally discovered due to its sensitivity towards the mTOR inhibitor rapamycin, responds to extracellular growth factor signaling, WNT signaling, and nutrient abundance via glucose and amino acid-triggered signaling. Downstream effectors of mTORC1 include autophagy, mitochondrial metabolic function, protein synthesis, and ribosome biogenesis. mTORC2, initially discovered as a rapamycin-insensitive complex of mTOR, responds to insulin, growth factor signaling, and inflammatory signaling such as tumor necrosis factor-alpha, with its downstream effectors being Akt, a key serine/threonine kinase that functions in cell division …

Characterization Of Biodistribution Of Transferrin And Receptor Binding Mechanism By Mass Spectrometry, Hanwei Zhao

Doctoral Dissertations

Protein-based therapeutics have emerged as a key driver of rapid growth in drug development pipelines. However, developing such protein drugs is not straightforward in most cases, the existence of physiological barriers greatly restricts the efficient delivery of many therapeutic molecules, and therefore limits their clinical applications. A promising way to address this challenge takes advantage of certain transport protein which can effectively across and enhance the permeability of these barriers, such as transferrin (Tf) which can be internalized by malignant cells and cross physiological barriers via transferrin receptor (TfR)-mediated endocytosis and transcytosis. However, developing such products is impossible without successfully …

Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham

Doctoral Dissertations

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into several functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Nine TPR-containing proteins have been shown to localize to the ER and control protein organization and …

Bubble Lab Exercise, Peter Beltramo

Science and Engineering Saturday Seminars

The cell membrane is a ubiquitous component in mammalian cells which control many vital biological functions. It consists of a phospholipid bilayer with embedded protein molecules which serve to transport molecules between the interior and exterior of the cell. Understanding what makes cell membranes so important and how they function requires concepts from physics, chemistry, and of course biology, but it is difficult to learn and conceptualize the structure and function of membranes due to their nanoscopic size and dynamic nature which can’t be properly appreciated in a static textbook. This activity draws analogies between the chemistry and structure of …

Rna-Seq And Mechanistic Enzymology Confirm Rna Self-Templated Extension By T7 Rna Polymerase And Suggest Novel Approaches Towards Improved In Vitro Rna Synthesis, Yasaman Gholamalipour

Doctoral Dissertations

Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyzed in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirmed that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. …

The Spatial Organization Of Mycobacterial Membrane, Julia Puffal

Doctoral Dissertations

Mycobacteria comprises a large group of organisms including the pathogenic species Mycobacterium tuberculosis, the causative agent of tuberculosis. A fast- growing saprophytic member of this genus, however, Mycobacterium smegmatis, is oftentimes used as a model organism for the pathogenic species. With a unique cell envelope architecture and unconventional polar growth, spatial coordination of cell envelope biosynthesis is vital for proper assembly of this complex structure. Here, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain (IMD), a spatially distinct region of the plasma membrane with diverse functions. …

Probing Apoptotic Caspase Allostery And Exosite Interactions For Alternative Regulation, Derek J. Macpherson

Doctoral Dissertations

Programmed cell death, or apoptosis is a critical homeostatic pathway that monitors the balance of cell life and death. Apoptosis is regulated by a class of enzymes known as the cysteine aspartic proteases, or the caspases. The 12 human caspases that play important roles in the progression and regulation of apoptosis and inflammation. Caspases are tightly regulated by numerous factors including enzymatic activation, post-translational modifications, metal ligand binding, and protein modulation. Aberrant caspase activation and regulation has been implicated in the progression of numerous diseases such as proliferative diseases and neurodegeneration. The deeply entwined nature of caspases and apoptosis makes …

Building The Outer Membrane Protein G (Ompg) Nanopore Library: From The Discrimination Of Biotin-Binding Proteins In Serum To Resolving Human Carbonic Anhydrase From Human Red Blood Cells, Bib Yang

Doctoral Dissertations

The use of pore-forming proteins (PFPs) in nanopore sensing has been fruitful largely due to their nanoscale size and the ease with which protein nanopores can be manipulated and consistently reproduced at a large scale. Nanopore sensing relies heavily on a steady ionic current afforded by rigid nanopores, as the change in current is indicative of analyte detection, revealing characteristics of the analyte such as its relative size, concentration, and charge, as well as the nanopore:analyte interaction. Rigid PFPs have been used in applications such as DNA sequencing, kinetic studies, analyte discrimination, and protein conformation dynamics at the single-molecule level. …

Nascent Dna Proteomics Analysis Uncovers Dna Replication Dynamics In The Human Pathogen Trypanosoma Brucei, Maria Rocha Granados

Doctoral Dissertations

DNA is the substrate of many cellular processes including DNA replication, transcription and chromatin remodeling. These processes are coordinated to maintain genome integrity and ensure accurate duplication of genetic and epigenetic information. Genome-wide studies have provided evidence of the relationship between transcription and DNA replication timing. A global analysis of DNA replication initiation in T. brucei showed that TbORC1 (subunit of the origin recognition complex, ORC) binding sites are located at the boundaries of transcription units. Although recent studies in T. brucei indicate functional links among DNA replication and transcription, the underlying mechanisms remain unknown. In this study, we adapted …

Protein Degradation Regulates Phospholipid Biosynthetic Gene Expression In Saccharomyces Cerevisiae, Bryan Salas-Santiago

Doctoral Dissertations

Transcriptional regulation of most phospholipid biosynthetic genes in Saccharomyces cerevisiae is coordinated by inositol and choline. Inositol affects phosphatidic acid (PA) intracellular levels. Opi1p interacts physically with PA and is the main repressor of the phospholipid biosynthetic genes. It is localized in the endoplasmic reticulum (ER) bound to the ER membrane protein Scs2p. When PA levels drop, Opi1p is translocated into the nucleus repressing most phospholipid biosynthetic genes. The OPI1 locus was identified in a screen looking for overproduction and excretion of inositol (Opi^-). Opi^- mutants are generally associated with a defect in …

Enhancing Nanopore Based Biosensening Technology Using Pore Forming Proteins, Christina M. Chisholm

Doctoral Dissertations

Pore forming proteins (PFPs) are membrane channels that are essential for various biological processes. For example, some PFPs act as gatekeepers of the cell, controlling the traffic of ions and macromolecules flowing into and out of cells; while others are involved in causing cell death (Reiner et al., 2012). Our fundamental understanding of PFPs determines our ability to employ these proteins for use in biomedical research and nanopore technology. Given their nanoscale dimensions, reproducibility and functionality these PFPs are widely used in the growing field of nanopore technology, particularly nanopore sensing (Reiner et al., 2012; Feng et al., 2015). These …

Identifying Functional Components Of The Endoplasmic Reticulum Quality Control And Degradation Factor Edem1, Lydia Lamriben

Doctoral Dissertations

The ER Degradation-Enhancing Mannosidase-Like protein 1 (EDEM1) is a critical endoplasmic reticulum (ER) quality control factor involved in identifying and directing non-native proteins to the ER-associated protein degradation (ERAD) pathway. However, its recognition and binding properties have remained enigmatic since its discovery. Here we provide evidence for an additional redox-sensitive interaction between EDEM1 and Z/NHK that requires the presence of the single Cys on the α-1 antitrypsin ERAD clients. Moreover, this Cys-dependent interaction is necessary when the proteins are isolated under stringent detergent conditions, ones in which only strong covalent interactions can be sustained. This interaction is inherent to the …

Applications Of Drosophila Melanogaster In Food Science Research, Phoebe Beverly Chen

Doctoral Dissertations

Drosophila melanogaster is a versatile model organism that provides several unique features, such as highly conserved disease pathways with humans as well as availability of environmental and genetic manipulations. Meanwhile, there is increasing interest in the potential role of bioactive food components, epigallocatechin gallate (EGCG) and conjugated linoleic acid (CLA), in metabolic research. However, there is limited knowledge on the sex-dependent effects of EGCG and CLA on energy metabolism. In this study, Drosophila melanogaster was used to investigate the sex-dependent effects of EGCG and CLA with respect to energy metabolism, including body fat, locomotion, and their key metabolic regulators. We …

The Interplay Between Polarity Regulators, Calcium, And The Actin Cytoskeleton During Tip Growth, Carlisle Bascom Jr

Doctoral Dissertations

Plant cell growth is a meticulously regulated process whereby the cell wall is selectively loosened to allow for turgor-pressure driven expansion. The rate of expansion must equal delivery of new material, or the cell will lyse. In many plant cells, this process happens diffusely around the cell. However, a number of plant cells have anisotropic shapes that require exquisite spatial control of secretion. One simple example of anisotropic patterning is tip growth; highly polarized cell expansion utilized by pollen tubes, root hairs, and moss protonemata. Investigating the role various molecules have in tip growth sheds light on how plant cells …

Feronia-Related Receptor Kinase 7 And Feronia And Their Role In Receiving And Transducing Signals, David Vyshedsky

Masters Theses

Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter …

Exploring The Influence Of Pkc-Theta Phosphorylation On Notch1 Activation And T Helper Cell Differentiation, Grace Trombley

Masters Theses

The T cell-specific kinase, Protein Kinase C theta (PKCq) is essential to T cell activation and differentiation. PKCq integrates T cell receptor (TCR) and CD28 signaling, and ultimately activates transcription factors necessary for full T cell activation, proliferation, survival, and differentiation into T helper (Th) subsets. Th1, Th2, Th17 and Treg cells compose the four major lineages of T helper cells, differentiated from CD4 T cells, and each have different requirements for PKCq.

PKCq, itself, is regulated through phosphorylation of specific resides, including tyrosine (Y)90 and threonine (T)538. Following T cell stimulation, PKCq is phosphorylated on Y90 by the kinase, …

Investigating The Role Of Rna-Binding Protein 5 In The Life Cycle Differentiation Of Trypanosoma Brucei, David Anaguano Pillajo

Masters Theses

Trypanosomatid parasites such as Trypanosoma brucei have unusual mechanisms of gene expression including polycistronic transcription, mitochondrial RNA editing and trans-splicing. Additionally, these protists rely mainly on post-transcriptional regulation where RNA-binding proteins (RBP) have shown to play a major role. RBP6 and RBP10 are two examples of RBPs that play crucial roles in procyclic and bloodstream form parasites differentiation respectively, by post-transcriptional regulation. Over-expression of RBP6 is enough to promote differentiation into metacyclic trypomastigotes that are infective to mice. However, continuous expression is required, and this pattern does not reflect the natural expression in the tsetse fly or the influence of …

Characterization Of She1 Spindle Role Using Ceullular, Biochemical, And Biophysical Methods, Yili Zhu

Doctoral Dissertations

During development, metaphase spindles undergo large movement and/or rotation to determine the cell division axis. While it has been shown that spindle translocation is achieved by astral microtubules pulling and/or pushing the cortex, how metaphase spindle stability is maintained during translocation remains not fully understood. In budding yeast, our lab has previously proposed a model for spindle orientation wherein the mitotic spindle protein She1 promotes spindle translocation across the bud neck by polarizing cortical dynein pulling activity on the astral microtubules. Intriguingly, She1 exhibits dominant spindle localization throughout the cell cycle. However, whether She1 has any additional role on the …

Extrinsic And Intrinsic Factors In Liver Development, Amrita Palaria

Doctoral Dissertations

Liver is the largest internal organ of the human body. It performs a multitude of functions. Therefore, it is provided with a huge regenerative capacity however, because of the same reason it is also prone to various diseases. Hence, it is essential to understand liver development in order to understand liver regeneration and liver diseases to provide better therapeutic targets and solutions. Liver development is orchestrated by a variety of intrinsic and extrinsic factors. The major focus of this dissertation thesis is to elucidate the role of BMP signals and YY1/VEGFA regulated signals in liver development. Liver organogenesis initiates with …

Examining Shsp-Substrate Capture And Chaperone Network Coordination Through Cross-Linking, Keith Ballard

Doctoral Dissertations

Small heat shock proteins (sHSPs) and related α-crystallins are virtually ubiquitous, ATP-independent molecular chaperones linked to protein misfolding diseases. They comprise a conserved core α-crystallin domain (ACD) flanked by an evolutionarily variable N-terminal domain (NTD) and semi-conserved C-terminal extension/domain (CTD). They are capable of binding up to an equal mass of unfolding protein, forming large, heterogeneous sHSP-substrate complexes that coordinate with ATP-dependent chaperones for refolding. To derive common features of sHSP-substrate recognition, I compared the chaperone activity and specific sHSP-substrate interaction sites for three different sHSPs from Arabidopsis (At17.6B), pea (Ps18.1) and wheat (Ta16.9), for which the atomic solution-state structures …

The Molecular Basis Of Caspase-9 Inactivation By Pka And C-Abl Kinases, Banyuhay Paningbatan Serrano

Doctoral Dissertations

Caspases are the cysteine proteases that facilitate the fundamental pathway of programmed cell death or apoptosis. The activation and function of these powerful enzymes are tightly regulated to ensure the faithful execution of apoptosis and prevent untimely cell death. Many deadly human diseases such as cancer, neurodegeneration and autoimmune disorders have been associated with defective activation and faulty regulation of caspases. As such, caspases are considered as attractive drug targets, which when properly controlled, can lead to effective therapeutics for apoptosis-related diseases. Thus, comprehensive investigations of the structure, function and regulation of caspases are necessary to understand the complex mechanisms …

Clpxp Functions In Caulobacter As A Universal And Species-Specific Protease, Robert Vass

Doctoral Dissertations

Proteolysis shapes many aspects of cellular survival, including protein quality control and cellular signaling. Powered proteolysis couples ATP hydrolysis with a degradation force that actively probes and interrogates the protein population. ClpXP, exemplifies a conserved two-part protease system charged with powered proteolysis. This protease exists as a regulatory element (ClpX), and a compartmentalized, self-contained peptidase element (ClpP). In Caulobacter crescentus, ClpXP degradation plays a crucial role maintaining proteins that exhibit proper activity, and also triggers the start of cellular differentiation. Substrate elimination requires shared aspects of the protease from both quality control and precision protein destruction functions. Here, the regulatory …