Albert Einstein College of Medicine (AECOM)
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Albert Einstein College of Medicine is a research-intensive medical school. For more than 60 years, our diverse faculty and staff have set the standard for excellence in medical and graduate education and patient-centered clinical care, and have made major contributions to scientific research enhancing human health in our communities and beyond. Our mission is to prepare a diverse body of students to become knowledgeable, compassionate physicians and innovative scientific investigators, and to create new knowledge.
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Item Metadata only 15q11.2 disease locus: From cognition to molecular(ProQuest Dissertations & Theses, 2016) Woo, Young JaeA large number of copy number variants (CNVs) associated with clinically defined psychiatric disorders show incomplete penetrance and variable expressivity. To better understand mechanisms and work towards personalized interventions, my research focused on investigating the intermediate brain and behavior phenotypes associated with BP1-2 deletion at 15g11.2, a multi-genic region that increases risk for schizophrenia and epilepsy. We have recruited genetically defined research subjects through social media and used online tools to perform cognitive assessments. We find that BP1-2 deletion carriers, but not their non-carrier family members, show domain specific cognitive impairments. Profound deficits in grammatical reasoning, arithmetic tasks, and visuo-spatial working memory are observed despite normal performance on non-verbal tasks.;Towards mechanisms, we looked for relationships between quantitative measures of brain structure obtained by MRI and common variants within BP1-2 region. After correcting for multiple comparisons, we observed an association between a variant 2 kb upstream of cytoplasmic FMR1 interacting protein 1 gene (CYFIP1) and the left supramarginal gyrus (lh.SMG), a brain region implicated in schizophrenia and language processing. Separate analyses in an independent cohort of 2622 typically developing adults showed an association between Ih.SMG and this same variant identified in our discovery cohort. Genotype at this Single Nucleotide Polimorphism (SNP), as well as seven linked SNPs, showed a significant correlation with CYFIP1 mRNA levels in human brain. In an attempt to understand how genetic variation in this region might come to impact CYFIP1 expression, we looked for transcription factor binding sites predicted to show allele specific binding. Strikingly, these investigations revealed that one of the seven CYFIP1 regulatory variants we identified is predicted to show allele specific binding for FOXP2, a transcription factor well known for its role in language. Separate analyses showing a strong correlation between FOXP2 and CYFIP1 levels in human brain (r 2=0.36), is consistent with FOXP2 being a key regulator of CYFIP1.;Genomic imbalance at BP1-2 region increases disease risk for a number of neurodevelopmental disorders and may be responsible for variety of clinically relevant traits. We performed online-based cognitive assessment and found a specific role of BP1-2 deletion in language processing and arithmetic tasks. Our work also shows the feasibility of using online tools to recruit populations of interest located worldwide and study disease-related quantitative traits that could be used in clinical trials. Our neuroimaging work points toward an importance of CYFIP1 in lh.SMG development, an effect that may be mediated by altered binding of the language-implicated transcription factor FOXP2. This work demonstrates the usage of neuroimaging in disease gene detection.Item Metadata only A 17th-century “Jewish” medical diploma(Rabbinical Council of America, 2022-07-24) Reichman, EdwardItem Metadata only 3,3'-diindolylmethane and genistein alter the effects of estrogen on prostate cancer cells(ProQuest Dissertations & Theses, 2009) Smith, SunyataEvidence suggests that 17beta-estradiol (E2) contributes to the risk of prostate cancer (PCa), whereas the phytochemicals genistein from soy and 3, 3'-diindolylmethane (DIM), derived from indole-3-carbinol (13C) in cruciferous vegetables, decrease the risk of prostate cancer (PCa). This study examined the potential of these phytochemicals to reduce the adverse effects of E2 on PCa. In LNCaP PCa cells (E2 sensitive) DIM decreased E2-induced proliferation. Genistein increased proliferation at low concentrations and decreased proliferation at higher concentrations; DIM abolished the increased proliferation by genistein. The E2 stimulation in LNCaP cells was consistent with dependence on the androgen receptor (AR) as was evidenced by inhibition of E2-induced proliferation with the anti-androgen casodex (CSDX), E2 stimulation of an androgen response element (ARE) luciferase reporter, and E2 stimulation of prostate specific antigen (PSA) protein expression. Both genistein and DIM abrogated the E2 stimulation of PSA. Genistein and DIM altered major E2 metabolism pathways in LNCaP and PC-3 (E2 insensitive) PCa cells by increasing expression of the 2-hydroxylation enzyme cytochrome P450 1A1 (CYP1A1) and the O-methylating enzyme catechol-o-methyltransferase (COMT) as determined by real time RT-PCR. The increase in COMT mRNA only occurred when the combination of DIM and genistein (151mumol/L) was used. Quantitation by mass spectrometry indicated an increase in 2-hydroxyestradiol (2-OHE2) and a decrease in 16alpha-hydroxyestrone (16alpha-OHE1), a result that should result in less estrogenicity and increased amounts of the anti-cancer metabolite 2-methoxyestrone. Furthermore, apoptosis studies in LNCaP and PC-3 cells that were treated with or without E2 in the presence of DIM and/or genistein revealed that estrogen significantly enhances DIM and genistein induced apoptosis. The enhancement of apoptosis by estrogen also occurred in MCF-7 breast cancer cells and C3 3A cervical cancer cells. Our conclusions are that DIM and genistein decrease effects of E2 that have the potential to promote PCa.Item Metadata only A biosensor of S100A4 metastasis factor activation: Inhibitor screening and cellular activation dynamics(ProQuest Dissertations & Theses, 2008) Garrett, Sarah C.S100A4, a member of the S100 family of proteins, binds nonmuscle myosin-IIA in a calcium-dependent manner and regulates the monomer-polymer equilibrium of myosin-IIA filaments. S100A4 displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 plays a direct role in metastatic progression, due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. To examine S100A4 activity in living cells, we developed a fluorescent biosensor that reports on the Ca 2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor (Mero-S100A4) that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for in vitro and in vivo use. Biochemical analysis demonstrates that the calcium and myosin binding properties of Mero-S100A4 are similar to wild-type S100A4 and validate its use for a wide range of studies. This biosensor shows that in fibroblasts, localized activation of S100A4 at the cell periphery is observed during stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Live cell imaging also revealed S100A4 activation in cellular regions undergoing retraction. In MDA-MB-231 human breast cancer cells crawling into a wound, S100A4 activation was detected in regions protruding toward the wound with the highest activation localized to dorsal ruffles. Additionally, a screen against a library of FDA approved drugs with the Mero-S100A4 identified an array of phenothiazines as inhibitors of myosin-IIA associated S100A4 function. These drugs are the first reported inhibitors of S100A4 and provide the foundation for future drug development. Together these data demonstrate the utility of the Mero-S100A4 biosensor for both drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.Item Metadata only A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis(ProQuest Dissertations & Theses, 2002) Chiu, Raymond C. N.In mammalian cells the Golgi apparatus consists of a series of flattened cisternae localized to the perinuclear region of the cell. The Golgi undergoes extensive and reversible fragmentation into vesicles and tubules during mitosis; these reassemble into cisternae at cytokinesis. A protein complex that mediates post-mitotic Golgi re-assembly has been characterized by several laboratories and consists of at least four core, high molecular weight proteins: p115, GM130, GRASP65 and giantin. p115 has been demonstrated to play a key role in vesicle tethering and is required for maintaining the structural organization of the Golgi apparatus. Phosphorylation of GM130 disrupts its interaction with p115 and prevents vesicle tethering to the Golgi. This is thought to be the key event in effecting mitotic Golgi fragmentation.;In contrast to mitosis, the Golgi undergoes irreversible fragmentation during programmed cell death (apoptosis). A main goal of this thesis research was to determine if Golgi fragmentation during apoptosis and mitosis occurred via a similar mechanism. The data suggest that this is not the case. GM130 was not phosphorylated and caspases were found to cleave both p115 and GM130 during apoptosis. Unlike protein phosphorylation during mitosis, the irreversible nature of protein cleavage during apoptosis results in the permanent inhibition of vesicle reassembly into Golgi cisternae. Compared to control cells expressing wildtype p115, those expressing a caspase cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an N-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the 30 kDa C-terminal caspase-cleavage product of p115 induced Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Deletion analysis showed that expression of a core fragment comprising 102 amino acids of the 30 kDa polypeptide was able to translocate to the nucleus and induce apoptosis. Most significantly, in vivo expression of the C-terminal fragment in the presence of caspase inhibitors or upon co-expression with a cleavage-resistant mutant of p115 showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.;In addition to p115, other factors implicated in maintaining Golgi structure were also cleaved by caspases during apoptosis. Among them is the beta isoform of the phosphatidylinositol 4-phosphate 5-kinase (mPIP5KIbeta). Like p115, expression of the C-terminal caspase cleavage fragment of mPIP5KIbeta also resulted in its translocation into the nucleus and the induction of Golgi fragmentation and apoptosis. Expression of the full-length or the N-terminal cleavage fragment of mPIP5KIbeta had no effect on Golgi structure or cell survival. In sum, our data suggest that cleavage of a single substrate to effect multiple events may be a common theme in apoptosis. This idea emphasizes the efficiency in which the apoptotic program causes the ultimate demise of the cell.Item Metadata only A CEREBELLAR ROLE IN CONTROL OF THE GOLDFISH VESTIBULO-OCULAR REFLEX(ProQuest Dissertations & Theses, 1983) MICHNOVICZ, JON JOSEPHThe vestibulo-ocular reflex (VOR) stabilizes gaze by producing eye movements that compensate for head rotations. VOR gain is defined as eye velocity divided by head velocity. Three types of experiments were performed in goldfish (Carassius auratus) to investigate the cerebellar role in gain control, including cerebellar ablation, microstimulation, and extracellular recording, before and after training.;Cerebellectomy increased the VOR gain. An average gain of 0.86 (+OR-) 0.12 in untrained animals at 1/8 Hz increased to 1.50 (+OR-) 0.36 after cerebellectomy (N = 9). Cerebellectomy almost immediately abolished gain changes previously established by training. Both visual optokinetic reflexes and VOR suppression were unimpaired by cerebellectomy.;Cerebellar microstimulation (1-6 (mu)A) evoked vigorous nystagmus. The stimulated areas may include the normal output to the brainstem vestibular centers. Results indicate activation or inhibition of cerebellar neurons exerting inhibitory control over the ipsilateral vestibular nucleus. Eye movements reversed direction with currents of reversed polarity.;Extracellular unit recording was performed both in the cerebellum and in the brainstem. Four types of activity were distinguished among cerebellar units, described here as eye velocity, velocity-position, molecular layer interneuron, and Purkinje cell units. Some Purkinje cells were discovered to be sensitive to changes in gaze velocity, i.e., modulating peak-to-peak firing during VOR suppression. Six of these Purkinje cells were followed continuously for 30-90 minutes of gain reduction training. Substantial increases in unit sensitivity occurred: unit sensitivity increased by roughly 0.1 ips/deg/sec with each drop of 0.1 in the gain. Phase change were also observed in several units.;These data demonstrate that the goldfish cerebellum strongly influences the functioning of the VOR. Cerebellar neurons are shown to be sensitive to errors in the VOR gain, suggesting a role in measuring the amplitude and direction of necessary longer-term gain changes. Enhanced firing of cerebellar neurons during gain decreases suggests that the cellular events underlying motor learning may take place within the cerebellum. A model is proposed to account for the different responses seen among cerebellar neurons during training.Item Metadata only A characterization of caveolins/caveolae in cardiac and smooth muscle tissues(ProQuest Dissertations & Theses, 2004) Woodman, Scott E.To better understand the role of caveola/caveolins in electrically responsive cells we examined cardiac muscle and urogenital smooth muscle tissues in mice genetically engineered to be caveolin deficient.;Only Cav-3 is expressed in adult mouse cardiac myocytes. Caveolin-3 knock-out (Cav-3 KO) mouse hearts fail to express the Cav-3 protein and Cav-3 KO cardiac myocytes do not form caveolae. These hearts still express Cav-1 and Cav-2, corresponding to the caveolae formation in cardiac endothelium. Cav-3 KO hearts are hypertrophy and display a reduction in fractional shortening by gated cardiac MRI and transthoracic echocardiography. Histological analysis also show Cav-3 KO hearts to be hypertrophic with an increase in cellular infiltrates. Although the expression and membrane association of dystophin-glycoprotein complex (DGC) proteins remain unchanged, a DGC marker, alpha-sarcoglycan, is excluded from lipid raft/caveolar domains. Mitogen-Activated Protein Kinase activity is increased in Cav-3 KO hearts. These results suggest that Cav-3 generated caveolae in cardiac myocytes play a role in facilitating membrane signaling.;All caveolin family members are expressed in adult mouse urinary bladder. Cav-1 KO mouse urinary bladders fail to express Cav-1, show a near complete loss of Cav-2, with no significant change in Cav-3 expression. Cav-3 KO mouse urinary bladders fail to express Cav-3, but express Cav-1 and Cav-2 in normal amounts. Caveolae formation was only reduced in Cav-1 KO mouse urinary bladders, showing Cav-1 to be the primary caveolae-forming caveolin family member. Cystometric analysis of urinary bladder function within Cav-1 KO mouse urinary bladders show higher basal, threshold, and spontaneous pressure measurements as compared to wild-type controls. Histological analysis reveals urinary bladder smooth muscle cell hypertrophy in the Cav-1 KO mouse. Cav-1 KO bladder strips have a diminished contractile response to the muscarinic agonist carbachol and KCl membrane depolarization. These results suggest that Cav-1 generated caveolae may play an important role in the facilitation of urinary bladder smooth muscle cell contraction.Item Metadata only A Closer Look at Cell Invasion with Biosensors(ProQuest Dissertations & Theses, 2013) Moshfegh, YasminThe Rho family of small GTPases control cell motility and migration, and are understood to participate in the regulation of cancer metastasis. Specifically, Rac1 GTPase is overexpressed in several tumors. Invadopodia are F-actin-rich protrusions with proteolytic activity that are exclusive to invasive tumor cells, and are thought to be crucial for cellular invasion and metastatic phenotypes. Based on previous literature, I hypothesized that the Rho GTPase Rac1 functions in the invadopodia of tumor cells. To investigate, I developed a new, genetically-encoded intramolecular Rac1 FRET (Forster Resonance Energy Transfer) biosensor. This new sensor is a substantial improvement over previous-generation Rac1 biosensors because of its single-chain arrangement, ensuring an equimolar distribution of FRET donor and acceptor, and producing a more accurate readout. Additionally, this design maintains the correct C-terminal lipid modification of full-length Rac1, enabling proper interaction with upstream regulators.;I used this new biosensor to observe the real-time activation dynamics of Rac1 at a subcellular level in live cancer cells. The biosensor readout shows Rac1 activity is excluded from the core of invadopodia, up to the point when invadopodia disappear, suggesting that a lack of Rac1 activity is necessary for their maintenance, and Rac1 activation is involved in disassembly. Focally uncaging Rac1 at pre-formed invadopodia using a photoactivation approach confirmed this previously-unknown Rac1 function. I integrated these results with existing literature to build an invadopodia disassembly model, where a signaling cascade starts with the regulator TrioGEF activating Rac1, triggering kinase activity of p21-activated kinase (PAK1), which phosphorylates cortactin, causing a structural destabilization and invadopodia dissolution. This mechanism may be critical for the proper turnover of the invasive structures during invasion of tumor cells in vivo, where a balance of invasive proteolytic activity and locomotory protrusions must be carefully coordinated to achieve a maximally invasive phenotype.;As an additional project, I designed and developed a new biosensor for the tyrosine kinase Src, which is well known for being necessary in invadopodia formation but its activation dynamics have not been shown directly. The biosensor can be used in the future as a reporter for Src activity in live tumor cells.Item Metadata only A DIRECT DETERMINATION OF THE MINIMUM SIZE FOR A EUKARYOTIC SECRETORY PRECURSOR (PREPROINSULIN, TRANSLOCATION, SIGNAL SEQUENCE, ENDOPLASMIC RETICULUM, GENE FUSION)(ProQuest Dissertations & Theses, 1986) ESKRIDGE, ETTA MARYMost eukaryotic secretory proteins are synthesized as precursors containing a transient amino-terminal signal peptide of 20-25 amino acids. The signal peptide mediates binding of polysomes to the endoplasmic reticulum (ER) via a co-translational interaction with signal recognition particle (SRP) and a receptor in the ER membrane. SPR interacts with nascent precursors after about 70 amino acids have polymerized, thus the prediction is that any small secretory peptide would be synthesized as a precursor with a minimum size of 70-80 amino acids. This hypothesis was tested directly by investigating the biosynthesis of preproinsulin, a precursor of 116 amino acids, and a series of preproinsulin truncations which spanned the predicted minimum size.;Segregation and processing of preproinsulin were SRP dependent and strictly co-translational in vitro. Synchronized kinetic studies showed that membrane interaction of nascent preproinsulin occurred when about 60-70 amino acids had polymerized; molecules beyond this size were incapable of initiating a functional membrane interaction. A series of cDNA clones encoding preproinsulin-like peptides spanning the predicted minimum size of 70-80 amino acids were generated. Each clone encoded the preproinsulin signal peptide and at least a portion of the mature hormone. The clones were transcribed and translated in vitro in the presence of microsomal membranes. The results indicate that peptides of 64 and 78 amino acids, but not 45 amino acids, were able to initiate membrane binding with an efficiency almost equivalent to preproinsulin. Signal cleavage of the 64 amino acid peptide was impaired, however; this may reflect inefficient translocation of this peptide through the ER bilayer.;Chimeric genes containing portions of preproinsulin cDNA and the coding sequences for the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT) were also constructed. CAT was sequestered into microsomal vesicles when attached to the preproinsulin signal peptide and B chain. The signal sequence was removed efficiently and accurately, and the fusions were glycosylated at a cryptic Asn-Gln-Thr site in the CAT molecule. These data suggest that the truncations of preproinsulin contained all the information necessary for membrane binding, translocation and processing, implying that the 45 amino acid peptide is deficient only in meeting the minimum size requirement.Item Metadata only A dopamine-dependent signal in the nucleus accumbens invigorates reward- seeking behavior(ProQuest Dissertations & Theses, 2014) du Hoffmann, Johann FaustusDecades of research have implicated ventral striatal dopamine in conditioned appetitive behaviors. We began with the observation that if the ventral striatal dopamine signal is impaired so is the ability of experimental subjects to approach reward-associated goals. A large population of ventral striatal neurons exhibits excitation elicited by reward-predictive cues. Intriguingly, the magnitude of these excitations predicts the presence (or absence) of subsequent reward-seeking behavior. We hypothesized that ventral striatal cue-evoked excitation is facilitated by dopamine and that this is the neural substrate that mediates conditioned approach to reward-associated objects. However, there was no experimental method available to test this hypothesis. To overcome this limitation, we developed a novel approach that enables us to record from neurons and locally infuse pharmacological agents in the same brain nucleus of behaving rats. We extensively tested this system and demonstrated that this method facilitates highly predictable, replicable and quantifiable drug effects on recorded neurons. Next, we locally infused dopamine receptor antagonists while recording from ensembles of neurons in ventral striatum as trained rats responded to reward predictive cues. In this manner, we directly tested the standard model of basal ganglia function which predicts that dopamine has both excitatory and inhibitory effects on neurons mediated by activation of D 1-like and D2-like dopamine receptors, respectively. We show that the net effect of signaling through either dopamine receptor type is to facilitate excitation, not inhibition. We demonstrate that co-activation of both dopamine receptor families specifically contributes to cue-evoked excitation and that this is required for environmentally cued appetitive behavior. Additionally, we show that tonic activation of dopamine receptors by agonists promotes cued approach behavior and antagonists to the same receptors have the opposite effect. This bidirectional control of appetitive behavior by pharmacological modulation of ventral striatal dopamine signaling firmly establishes its important role in cued approach behavior. In conclusion, we demonstrate the neural mechanism by which ventral striatal dopamine promotes reward-seeking: dopamine facilitates vigorous short latency approach behavior by facilitating strong cue-evoked excitation in ventral striatum.Item Metadata only A functional genomics approach to elucidate the role of genome maintenance in human longevity(ProQuest Dissertations & Theses, 2014) Han, JeehaeThe genetic control of longevity and pro-longevity phenotypes is likely to be determined by subtle variations in many genes involved in multiple genetic pathways. The challenge is to identify the combinations of alleles that are associated with healthy aging and longevity, and to ascertain the functional relevance of positive associations by providing mechanistic insights. There is strong evidence that genome maintenance is a major "Longevity Assurance Pathway" because genetic defects in this pathway cause a shorter life span and premature aging in humans and mice. The goal of my project is to elucidate the role of genome maintenance in human longevity by testing the hypothesis that a cluster of protective genotypes in the genome maintenance pathways is necessary to achieve exceptional longevity. To address this hypothesis, I conducted a systematic functional genomics approach to discover "functional genetic variants" involved in genome maintenance that are either enriched (beneficial alleles) or depleted (deleterious alleles) in Ashkenazi Jewish families with exceptional longevity. For this purpose, I utilized targeted sequence capture and next generation sequencing to discover all possible genetic variation in the coding and regulatory regions of 397 candidate genes acting in genome maintenance pathways. I discovered novel, potentially functional variants, including rare missense and regulatory variants that are significantly enriched in centenarians as compared to controls. Also, gene-based rare variation analysis followed by pathway analysis indicated that double strand break repair genes were enriched with longevity-associated rare variants. Since it is important to directly test the impact of rare variants by functional analysis for their longevity association and biological significance, in vitro cell culture study to assess the functional relevance of the rare double SIRT6 variant in the same haplotype was performed. The results demonstrated that the double SIRT6 mutant increased SIRT6 deacetylase activity specifically on H3K56Ac but not H3K9Ac in cell-based assays.;This study presents the first large scale candidate gene association study on human longevity utilizing high-throughput sequencing. We discovered new longevity loci enriched with rare variants within the genome maintenance pathway that could potentially provide important insights into the molecular basis of human longevity.Item Metadata only A genetic analysis of NPC1 receptor activity in Ebola virus infection(ProQuest Dissertations & Theses, 2017) Biwas, RohanFiloviruses are enveloped, negative-sense, single-stranded RNA viruses that are the causative agents of numerous sporadic outbreaks of viral hemorrhagic fever diseases with case fatality rates up to 90%. Recently, the unprecedented 2013-2016 Ebola virus outbreak in West Africa re-emphasized the grave, epidemic potential that filovirus outbreaks can have on a global scale and resulted in renewed efforts to develop useful and effective therapeutics to combat these deadly diseases.;Filovirus infections require the presence of the intracellular, host protein Niemann-Pick Cl (NPC1). NPC1 is a large, multi-pass 13 transmembrane protein with three luminal domains (A, C, and I) that resides in late endosomes/lysosomes (LE/LY) and is involved in the cellular distribution of free cholesterol to post-LE/LY compartments. Mutations in NPC1 can cause Niemann-Pick Disease Type C (NPC), a rare and fatal inherited disease marked by the endosomal accumulation of free cholesterol and sphingolipids in the brain and other tissues.;Since the breakthrough identification of NPC1 as a critical entry receptor for filoviruses in 2011, it has been the focus of many studies aiming to clarify the filovirus entry mechanism through an investigation of the virus-host interaction. Additionally, because NPC1 is required for the entry of all filoviruses, it is an especially attractive target for antiviral therapeutics because disruption of NPC1 receptor activity could confer pan-filovirus protection.;NPC1 luminal domain C is necessary and sufficient to mediate binding to the EBOV surface glycoprotein (GP), which is responsible for mediating viral entry into host cells. NPC1 domain C has been shown to directly bind GP through the use of two membrane-distal loops that interact directly with the hydrophobic binding pocket of GP. Although it is well established that luminal domain C plays a major role in the virus-host interaction, multiple lines of evidence have indirectly implicated the other luminal domains in the filovirus entry pathway.;In this thesis, we employ a genetic screen using a library of NPC-causing NPC1 mutant proteins in order to investigate the full-length NPC1 protein for residues that may play a role in filovirus entry. Our work identified three mutations, P401L (domain C); Y890C (domain I); and Y899D (domain I), that constitute a new class of NPC1 proteins -- ones that reduce EBOV infection without abrogating NPC1-GP binding. Recently published structures of NPC1 show that the domain C mutant, P401L, exists at an interface between the luminal domains C and I. Furthermore, our work highlighted specific residues, Y890C and Y899D, that are located outside of domain C that influence NPC 1 receptor activity. Together, these findings build on the notion that other luminal domains are, in fact, important in NPC1 receptor activity.Item Metadata only A genetic and molecular analysis of two Drosophila meiotic mutants encoding kinesin-like proteins(ProQuest Dissertations & Theses, 1992) Knowles, Brenda BrodeurThe nod, no distributive disjunction and ncd, non-claret disjunctional are female-specific, recessive meiotic mutations in Drosophila melanogaster. Mutations at either locus show high frequencies of chromosome nondisjunction at meiosis I and both loci have been shown to encode kinesin-like proteins. Unlike the ncd mutation, which affects all chromosome pairs, nod only affects disjunction of nonexchange chromosomes. This report describes experiments that seek, through second-site noncomplementation analysis and in situ hybridization experiments to define the relationship between these genes.;Although both the nod and ncd mutations are fully recessive, females doubly heterozygous for nod and ncd mutations were found to have levels of X and fourth chromosome nondisjunction 6-to-35 fold above those observed in control females. We infer that ncd is a dominant enhancer of nod. In situ hybridization data reveal that these transcripts have largely overlapping expression patterns during oogenesis. We propose that the relationship between the nod and ncd kinesin-like proteins is not one of direct structural interaction, but rather one defined by the temporal nature of the meiotic process.Item Metadata only A genetic approach to H1 histone function(ProQuest Dissertations & Theses, 1996) Sirotkin, Allen MarkThe mouse H1 histone gene family encodes a group of at least seven basic protein variants that bind to DNA and facilitate the formation of higher order chromatin structures. Interestingly, among the histones the H1 class displays the most complex pattern of subtypes including differentiation-specific and tissue-specific members. The large number and different patterns of expression among the H1 subtypes suggests that these H1 variants are in part responsible for the wide variations in chromatin condensation that exist within the genome and in different cell types. However, proof for a functional difference for the H1 variants is not available.;The H1{dollar}\sp\circ{dollar} subtype accumulates in several tissues during the first few weeks of postnatal mouse development and is found at highest concentrations in terminally differentiated tissues with a low rate of cell proliferation. In addition, we have shown that the H1{dollar}\sp\circ{dollar} gene is very rapidly induced at the transcriptional level during the precommitment period of mouse erythroleukemia (MEL) cell differentiation. To investigate the role of H1{dollar}\sp\circ{dollar} in mouse development, I have disrupted the single-copy H1{dollar}\sp\circ{dollar} gene by homologous recombination in mouse embryonic stem (ES) cells and transmitted this mutation to the mouse germ line. Surprisingly, mice lacking H1{dollar}\sp\circ{dollar} protein exhibited no apparent anatomical or phenotypic abnormalities and were born within the expected range predicted by Mendelian genetics. Furthermore, both male and female homozygotes were fertile and could be bred to produce additional homozygous progeny. Northern and Western analysis confirmed the absence of H1{dollar}\sp\circ{dollar} RNA and protein respectively in the mutant animals. Chromatin from H1{dollar}\sp\circ{dollar}-deficient animals showed no significant change in the relative proportions of other H1 subtypes or in the stoichiometry between linker histones and nucleosomes, suggesting that other H1 histones can compensate for the deficiency in H1{dollar}\sp\circ{dollar} by occupying sites normally containing H1{dollar}\sp\circ.{dollar} These results argue that H1{dollar}\sp\circ{dollar} function is dispensable for normal embryogenesis and demonstrate that other members of the H1 histone family can compensate for the loss of H1{dollar}\sp\circ.{dollar} Mice homozygous for disruptions at the H1c and H1e loci have also been generated and are presented in the thesis as well.Item Metadata only A genetic approach to the functions of specific H1 linker histones in mammalian spermatogenesis(ProQuest Dissertations & Theses, 1999) Lin, QingcongHistones are small, basic proteins found in abundance in most eukaryotic cells. The H1 histones bind the linker DNA between nucleosome core particles and facilitate the packaging of chromatin into the 30-nm fiber and higher order structures.;To investigate the role of the testis-specific linker Histone H1t in spermatogenesis, the H1t gene was disrupted by homologous recombination in mouse embryonic stem (ES) cells that were then used to generate mice that transmitted the disrupted H1t allele. Analysis of H1s in testis extracts from H1t(-/-) male mice showed that they were completely lacking H1t. These mice are fertile and produce normal numbers of mature sperm. The results indicate that despite the unique properties and expression pattern of H1t it is not essential for spermatogenesis.;Analysis of male germ cells from H1t -/- mice showed that the somatic H1a subtype predominated in chromatin. To determine the effect of creating a larger linker histone deficiency in chromatin than that established by the disruption of H1t alone, we disrupted the H1a gene in ES cells also containing a disrupted H1t allele. Targeting of the H1a gene in ES cells occurred both in cis and in trans to the modified H1t allele. Both strains of mutant mice are viable, appear normal, and have normal body weights and testis weights. Both strains also are fertile and have normal numbers of mature sperm.;HPLC analyses showed that the H1 to nucleosome ratio is the same in H1a(-/-) as in wildtype littermates. HPLC data also showed that the H1d and H1e subtypes preferentially substitute for the loss of H1a. The percentage of H1t in total H1s was not altered. This result indicates that H1t is able to replace H1c, H1d, and H1e, as effectively as it normally replaces H1a, Thus H1a, is not required for the H1stone protein transition from somatic H1s to H1t.;Analyses of germ cell chromatin in the H1at double mutants showed that the H1 to nucleosome ratio decreased by 25%, indicating that the mice cannot fully compensate for loss of 70% of their H1. The observed 25% decrease in the H1 to nucleosome ratio in the total germ cells of the double mutant implies that pachytene spermatocytes in the double mutant must have an H1 to nucleosome ratio 50% that of normal. Nevertheless spermatogenesis is normal in these mice. These results indicate that both H1a and H1t are not essential for normal spermatogenesis, suggesting that H1s may be partially functionally redundant, at least in spermatogenesis. (Abstract shortened by UMI.).Item Metadata only A genetic study of the anti-DNA associated idiotype 8.12: Analysis of 8.12(+) antibodies and autoantibodies, and characterization of the VlambdaII gene family(ProQuest Dissertations & Theses, 1993) Paul, ElahnaIndividuals with systemic lupus erythematosus (SLE) characteristically display high serum titers of autoantibodies directed against a variety of nuclear antigens. While SLE autoantigens typically include both proteins and nucleic acids, antibodies against double stranded DNA are the hallmark of this disease and have been implicated in its pathogenesis. 8.12 is an anti-DNA associated idiotype present in elevated titers in the serum of over 50% of patients with SLE and can constitute up to one third of a patient's anti-DNA antibodies. Generation and analysis of a panel of 8.12{dollar}\sp+{dollar} antibodies, both DNA binding and non-DNA binding, was undertaken to identify the genes which encode anti-DNA antibodies and the structures that impart anti-DNA specificity. In conjunction with RFLP studies of 8.12- encoding genes, such an analysis may elucidate the origins and the (dys)regulation of SLE anti-DNA autoantibodies. This thesis comprises three sections: (1) correlation of the 8.12 idiotypic family with the V{dollar}\lambda{dollar}II gene family and genetic mapping of the idiotope to a region near CDR1, (2) characterization of the V{dollar}\lambda{dollar}II gene family and determination of V{dollar}\lambda{dollar}II and C{dollar}\lambda{dollar} polymorphisms in SLE, and (3) a structural and mechanistic analysis of two 8.12{dollar}\sp+{dollar} anti-dsDNA antibodies, exploring their origins and evaluating their relationship to idiotypically and/or genetically homologous DNA and non-DNA binding antibodies. ftn*All degree requirements completed in 1992, but degree will be granted in 1993.Item Metadata only A guide for today’s perplexed? The changing face of Maimonidean scholarship(Academic Studies Press, 2023) Shatz, David.; Eleff, Zev; Seidler-Feller, ShaulEmet le-Ya‘akov comprises a collection of essays celebrating the career and achievements of Rabbi Dr. Jacob J. Schacter, who has served the American and international Jewish community with distinction in his roles as a synagogue rabbi, university professor, and public intellectual. These articles, like the honoree, recognize the importance of both history and memory, emphasize the necessity of accuracy in historiography, and do not shy away from inconvenient truths. They are divided into three categories that help frame the discussion around “facing the truths of history”: Textual Traditions, Memory and Making of Meaning, and (Re)Creating a Usable Past. The volume also includes a brief sketch of Schacter’s life and work and a bibliography of his publications.Item Metadata only A KAPtivating protein: Insights into the function of AKAP-KL(ProQuest Dissertations & Theses, 2002) Becker, Laren SethClassical A kinase anchor proteins (AKAPs) preferentially tether type II protein kinase A (PKAII) isoforms to sites in cytoskeleton and organelles. It is not known if distinct proteins selectively sequester regulatory (R) subunits of type I PKAs, thereby diversifying functions of these critical enzymes. In C. elegans, a single type I PKA mediates all aspects of cAMP signaling. I have discovered a cDNA that encodes a binding protein (AKAPCE) for the regulatory subunit (RCE) of C. elegans PKAICE. AKAPCE is a novel, highly-acidic, RING finger protein composed of 1,280 amino acids. It binds RI-like R CE with high affinity and neither RIIalpha nor RIIbeta competitively inhibits formation of AKAPCE·RCE complexes. The RCE binding site was mapped to a segment of 20 amino acids in an N terminal region of AKAPCE. Three large aliphatic amino acids (L236,I248,L252) in the tethering domain of AKAPCE (residues 236--255) are crucial for ligation of RCE. Their sidechains apparently generate a precisely-configured hydrophobic binding pocket that accommodates an apolar surface on RCE dimers. Basic residues (H254R255K 256) at the C terminus of the tethering site set an upper limit on affinity for RCE. A central dipeptide (F243S 244) contributes critical and distinctive properties of the tethering site. S244 is essential for selective binding of RCE and exclusion of RII isoforms. The aromatic hydrophobic character of F 243 ensures maximal RCE binding activity, thereby supporting a "gatekeeper" function of S244. Substitution of F243S244 with LV generated an RII-specific AKAP. RCE and RII subunits contain similar dimerization domains. AKAP-binding domains of RCE (residues 23--47) and RII differ markedly in size, amino acid sequence and docking specificity. Four hydrophobic residues (C23, V27, I32, C44) in RCE are crucial for avid binding with AKAPCE, whereas sidechains from L20,L35,V36,I 40 and I41 have little impact on complex formation. Y26 is embedded in the docking domain, but its aromatic ring is required for RCE-RCE dimerization. Residues 236--255 in AKAPCE also constitute a binding site for mammalian RIalpha. RIalpha (PKAIalpha) is tightly sequestered by AKAPCE in vitro (KD ∼ 10 nM) and in the environment of intact cells. AKAPCE·RCE and AKAPCE·RIalpha complexes accumulate in intact cells. Thus, the tethering domain of AKAP CE provides a molecular module for manipulating intracellular localization of RI and elucidating functions of anchored PKAI in eukaryotes.Item Metadata only A LIGHT-MICROSCOPIC AND ELECTRON-MICROSCOPIC STUDY ON NUCLEOLAR CONFIGURATIONS IN CELLS OF THE KANGAROO RAT: DIPODOMYS MERRIAMI(ProQuest Dissertations & Theses, 1973) MELKER, RICHARD JOELItem Metadata only A link between TORC1 and CK2 in S. cerevisiae via the Cdc-like kinase Knsl(ProQuest Dissertations & Theses, 2016) Sanchez-Casalongue, Manuel EduardoThe regulation of energy utilization in response to changing intracellular and extracellular conditions is key for survival. The Target of Rapamycin Complex 1 (TORC1) plays a central role in signaling nutrient availability, stress conditions and cellular energy levels to downstream anabolic and catabolic pathways in order to promote cell growth and prevent cell death. TOR kinase activity balances the level of protein synthesis and protein synthetic capacity (i.e. ribosome biogenesis) according to the needs of the cell. Ribosome biogenesis is an energetically costly process that accounts for over 80% of the transcriptional activity in growing cells and involves the coordinated action of all three nuclear RNA polymerases (pols I-III). The regulation of ribosome and tRNA synthesis is therefore a central hub for cellular energy management. Additionally, the dysregulation of this process is highly correlated with multiple cancers, making this system attractive for the development of new anti-cancer therapies.;Previously, our laboratory identified a new role for the cyclin-like kinase Knsl and the GSK3 kinase Mckl in the regulation of ribosome and tRNA synthesis downstream of TORC1. The first part of this thesis describes the design of a chemical-genetic screen identifying likely new roles for Knsl and Mckl in the regulation of vesicle-mediated transport, the control of cell size and polarity, and a link to protein kinase CK2. The second part of this thesis focuses on understanding the connection between TORC1 and CK2. Protein kinase CK2 is a constitutively active pro-growth kinase involved in cell cycle progression, cell proliferation and transcription and which contributes to cancer in higher organisms. We present evidence of in vivo phosphoregulation of its Ckbl subunit under nutrient limitation and cellular stress, and show that Knsl mediates this phosphorylation in all tested conditions. To query CK2 regulatory mechanisms we carried out co-immunoprecipitations of its subunits and glycerol gradient fractionations of whole cell extracts and found no evidence for the dissociation of CK2 subunits under stress. Instead, chromatin immunoprecipitation assays support a model of CK2 regulation via the differential association of the holoenzyme at pol III genes, and suggest a role for Ckbl phosphorylation in this process.