Dissertations from the School of Arts and Sciences
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- Alcohol consumption impairs protein trafficking: mechanism, consequences and possible treatment
- Efforts have been made to understand alcohol-associated liver disease (ALD) since the time alcohol was proved to independently cause liver injury. Although significant progress has been achieved in describing the clinical presentation of the disease, molecular mechanisms involved in the establishment of ALD to end-stage liver injuries are still not fully understood. Chronic alcohol consumption is one of the prerequisites for the development of severe forms of ALD such as fibrosis and cirrhosis. In rodents’ liver and hepatic cells in culture, chronic alcohol exposure leads to enhanced protein acetylation and acetaldehyde adduction. Of the multitude of proteins known to be modified upon alcohol administration, tubulin is among the best studied. However, an open question is whether these modifications present in clinical samples. Both modifications have also been implicated in promoting alcohol-induced defects in protein trafficking, but whether they do so directly is another open question. Due to limited knowledge of underlying molecular details, there is no effective treatment for ALD to this date. We induced tubulin hyperacetylation and acetaldehyde adduction in absence of alcohol and examined selective hepatic protein trafficking steps. We first confirmed that tubulin was hyperacetylated and acetaldehyde-adducted in livers from alcohol-exposed individuals. Then, we induced acetylation by overexpression of a tubulin-specific acetyltransferase, αTAT1, and adduction by directly adding acetaldehyde to WIF-B cells. Both αTAT1 overexpression and acetaldehyde treatment significantly impaired both plus-end (secretion) and minus-end (transcytosis)-directed microtubule-dependent trafficking and clathrin-mediated endocytosis. Each modification led to similar levels of impairment as observed in alcohol-treated cells. Levels of impairments by either modification showed no dose-dependence and no additive effects suggesting that sub-stoichiometric tubulin modifications lead to altered protein trafficking and that lysines are not selectively modified.To investigate effective intervention for ALD, we examined a dietary mimetic, spermidine, for its hepatoprotective properties. Because alcohol consumption is associated with oxidative stress-induced liver injury and pro-inflammatory responses, spermidine is likely to protect ALD by its antioxidant and anti-inflammatory characteristics. In this study, we used a two-hit, chronic alcohol and acute lipopolysaccharide (LPS)-induced mouse model of liver injury. We determined that spermidine administration prevented alcohol and LPS-induced increases in liver injury using plasma ALT and histology as a readout. As predicted, spermidine also prevented alcohol and LPS-induced oxidative stress by decreasing the levels of both reactive oxygen species and lipid peroxidation. Spermidine also prevented fibrosis by inhibiting alcohol and LPS-induced expression of pro-inflammatory cytokines, activation of hepatic stellate cells, and deposition of collagen. Together, our results indicate that spermidine is an antioxidant thereby conferring anti-inflammatory and anti-fibrotic effects associated with ALD., Biology, Cellular biology, Molecular biology, Biology, Degree Awarded: Ph.D. Biology. The Catholic University of America
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- Observed Interaction in Families of Adolescent Suicide Attempters
- Degree awarded: Ph.D. Psychology. The Catholic University of America, Attempted suicide during adolescence is disturbingly common. Prevention depends upon careful identification of factors that may contribute to its etiology and maintenance. Researchers have focused on a range of possible risk factors, including problems within the family. While promising, this research has suffered from a number of limitations, in particular an over-reliance on self-report methodology. The current study investigated family interactions of adolescent suicide attempters, using observational methods and a longitudinal design. Participants included 71 families of hospitalized attempters and 29 families of psychiatric controls. Families completed a variety of self-report measures as well as a videotaped problem-solving interaction task. Interactions were coded for a range of behaviors, including emotional validation and invalidation, problem-solving constructiveness, and problem-solving progress. It was expected that families of adolescent suicide attempters would display more negative behavior than families of hospitalized non-attempters, and that negative behavior within the suicide group would be related to individual factors such as psychopathology and beliefs about problem-solving. It was further expected that negative behavior at baseline would predict suicidal ideation and reattempted suicide during an 18-month follow-up period. There was at least partial support for each of the primary hypotheses. There were no significant differences in observed parent behaviors. However, adolescent attempters displayed significantly more emotional invalidation than psychiatric controls. Within the suicide group, negative beliefs about family conflict and problem-solving predicted observed negativity, for both parents and adolescents. In several cases, higher levels of adolescent psychopathology predicted more negative behavior as well. Finally, while parent behavior was not a significant predictor for subsequent adolescent suicidality, certain aspects of adolescent negativity predicted both reattempts and future suicidal ideation. Findings demonstrated that it is possible to observe distinct patterns of interaction in families of adolescent suicide attempters, and emphasized the value of a focus on adolescent, not just parent, behavior. Results suggested that adolescent attempters may have particular difficulty coping with affectively charged parent-adolescent conflict, and indicated that negative behavior (for both parents and adolescents) may be maintained by pessimistic cognitions. Results thus suggested important directions for future research, as well as possibly fruitful avenues for treatment and prevention., Made available in DSpace on 2012-04-02T15:43:03Z (GMT). No. of bitstreams: 1 Aiken_cua_0043A_10145display.pdf: 1794559 bytes, checksum: 8556e14c8b466c2e61037c248d9530bb (MD5)
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- Thermoelectric Properties and Transport Mechanisms of Lead- and Lead-Yttrium Ruthenate Pyrochlores
- The scope of this research was to investigate thermoelectric properties of selected pyrochlores, mainly lead ruthenate (Pb2Ru2O6.5) and some derivatives as well as solid solutions with yttrium ruthenate (Y2Ru2O7). The properties are thermal- κ(T) and electrical conductivity σ(T) and the Seebeck coefficient S(T). First, all materials were synthesized by solid state reaction of oxide powders at high temperature, and all are p-type. Except Y2Ru2O7, all compounds are defect pyrochlores with oxygen deficiency. Thermoelectric properties were measured from 25 to 300ºC. Power factors and figures of merit have been calculated. In a second step, the focus was on understanding the experimental data based on the pyrochlore crystal structure, A- and B-lattice site occupancy, ligand and crystal fields and scattering mechanisms. Pb2Ru2O6.5 and derivatives are metallic; σ(T) decreased with increasing temperature but κ(T) was low and increased. This behavior relates to intrinsic defects such as oxygen vacancies and a stereo-chemically active 6s2 electron lone-pair on Pb2+. The low and increasing κ(T) is typical of glass-like behavior. All experimental data for σ(T) and κ(T) were analyzed for the underlying scattering mechanisms. σ(T) and κe(T) varied with T -1/2 and T 1/2, respectively, implying impurity scattering at T >>TF. The contribution of electronic thermal conductivity κe to the total κ(T) increased with increasing temperature. The lattice contribution κL decreased with T -1, suggesting 3-phonon (Umklapp) scattering and supporting the electron-impurity scattering mechanism. S(T) showed a maximum at 423 K due to generation of minority electrons at high temperature.As to pyrochlore Pb2Ru2O6.5-Y2Ru2O7 solid solutions: With increasing yttrium content a metal-to-insulator transition (MIT) was discovered and explained by the Mott-Hubbard model. For S(T) and κ(T) the same was found as above. Scattering mechanisms were different for metallic, intermediate, and for insulator/semiconductor behavior of the pyrochlores. Scattering mechanisms were identified as impurity scattering on the metallic side of the MIT (Pb2-xYxRu2O6.5+z, x<0.2) and as Mott-Variable-Range-Hopping (σ(T) varies with exp(-1/T)1/4) on the semiconductor/ insulator side of the MIT (Pb2-xYxRu2O6.5+z, x≥1.5). In the transition zone the process of data analysis allowed for several scattering processes. There could be a combination of scattering mechanisms., Physics, Materials Science, Quantum physics, Glass-like thermal conductivity, Metal-insulator transition, Mott-Hubbard transition, Pyrochlore, Scattering mechanisms, Thermoelectric, Physics, Degree Awarded: Ph.D. Physics. The Catholic University of America
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- Return currents in solar flares: Theory and observations
- Electron beams are thought to be accelerated during a solar flare by conversion of magnetic energy into kinetic energy. These energetic beams are accelerated in the corona and stream both toward the interplanetary medium and toward lower (and denser) layers of the solar atmosphere. They constitute a current which is neutralized by a co-spatial return current. Several questions arise about the beam/return current system, and its coupling with the plasma in which it propagates. What is the magnitude of the beam current? Is this beam stable to the generation of current-driven instabilities? Does the return current heat the plasma through Joule heating? If so, where and how much energy is deposited in the solar atmosphere? In this thesis, we explore these questions, using a 1D model with a co-spatial return current to explain hard x-ray (HXR) spectral breaks. We study 19 flares observed by \texttt{RHESSI} (Ramaty High Energy Solar Spectroscopic Imager) with strong spectral breaks at energies around a few deka-keV, that cannot be explained by isotropic albedo or non-uniform ionization alone. We identify these breaks at the HXR peak time, but we obtain 8 second-cadence spectra of the entire impulsive phase. Electrons with an initially power-law distribution and a sharp low-energy cutoff lose energy through return-current losses until they reach the thick target, where they lose their remaining energy through collisions. The return current thick-target model (RCCTTM) and method of analysis are introduced in chapter 2. Our main results are summarized as follows: (1) The RCCTTM provides acceptable fits for spectra with strong breaks (chapter 3). (2) Limits on the plasma resistivity are derived from the fitted potential drop and deduced electron-beam flux density, assuming the return-current is a drift current in the ambient plasma. These resistivities are typically 2-3 orders of magnitude higher than the Spitzer resistivity at the fitted temperature, and provide a test for the adequacy of classical resistivity and the stability of the return current. (3) Using the upper limit of the low-energy cutoff, the return current is always stable to the generation of ion acoustic and electrostatic ion cyclotron instabilities when the electron to ion temperature is less than 9. (4) In most cases the return current is most likely primarily carried by runaway electrons from the tail of the thermal distribution rather than the bulk drifting thermal electrons. For these cases, anomalous resistivity is not required (chapter 4). In addition, we develop two steady-state numerical codes.One code self-consistently solves the propagation of the beam and return current including the contribution from runaway electrons in the thermal tail distribution, in the weak (sub-Dreicer) electric field regime. We find that when the return current is carried at least partially by runaway electrons, (1) the total energy deposited in the loop is reduced compared to the case where the return current is carried by the bulk thermal electrons, and (2) the return current electric field increases from the looptop to the thick target, if no beam electrons are thermalized (chapter 5).Recognizing the possibe importance of Coulomb collisions in the corona, we solve the energy loss equations taking into account Coulomb collisions and return current losses simultaneously, and obtain the spatial evolution of the electron distribution and X-ray emission along the loop. This is based on Emslie 1980 and Holman 2012 with updates on the classical resistivity in a partially ionized plasma from Sykora et al.2012. The importance of including collisional losses is increased for lower values of the injected electron low-energy cutoff, and if collisions are also taken into account, the flattening of the electron distribution is stronger lower in the loop (chapter 6)., Physics, Return current, Runaway electrons, Solar flares, X-rays, Physics, Degree Awarded: Ph.D. Physics. The Catholic University of America
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- Trauma Exposure and Mental Health for Children and Adolescents in a Nationally Representative Sample
- Research on trauma experienced by children and youths in the United States has been typically restricted to a specific trauma or population. An extensive, in-depth survey (National Survey of Children’s Exposure to Violence III; NATSCEV III) was administered to 4,000 participants across the United States between 2013 and 2014. The following studies conducted several secondary data analyses on the data gathered from the survey. First, using SPSS Version 27, descriptive statistics were conducted to examine overall rates of endorsed traumatic experiences, as well as mental health outcomes. Second, regression analyses were conducted to determine the relationship between trauma and posttraumatic symptoms in adolescents, especially considering categories of traumatic experiences, such as conventional crime, child maltreatment, and peer and sibling victimization. Last, moderation analyses were conducted to investigate potential moderators of that relationship, including variables such as ethnicity, household income, parenting style, and discipline. Results from these analyses indicated that bullying was the most commonly endorsed trauma for children and youths, which is concurrent with national data. Additionally, PTSD severity was found to be significantly predicted by number of traumas for adolescents. The household income of the adolescent’s residence and having a caregiver with an authoritarian parenting style were found to moderate the relationship between trauma and PTSD severity for adolescents. These results further our understanding of the rates of victimization and mental health outcomes for youth in the United States, as well as how the two are related and affected by other variables. This information is essential to understanding the additional challenges facing children and adolescents when regarding trauma, as well as helping clinicians and child welfare systems to promote resiliency in children and families. These results further lend themselves to the development and implementation of trauma-focused and evidence-based interventions, as well as aiding others in understanding the importance of trauma prevention, assessment, and treatment. , Clinical psychology, Adolescents, Children, Nationally Representative, Trauma, Psychology, Degree Awarded: Ph.D. Psychology. The Catholic University of America
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- Identifing the Amino Acid Residues Responsible for Molecular Diode Function in Pdr5, a Major Multidrug Efflux Pump
- Antimicrobial resistance (AMR) occurs when a microorganism gains resistance to antimicrobial agents that were originally designed to cure infections caused by these microorganisms. Overexpression of ATP-binding cassette (ABC) multidrug transporters is a mechanism of AMR, which acts by pumping the drugs out of the cell. ABC transporters are a superfamily of polytopic proteins that uses the energy of ATP hydrolysis to transport the substrates across the membrane against a concentration gradient. The typical ABC transporters consist of two transmembrane domains (TMDs), composed of six transmembrane helices (TMHs) that are connected by extracellular loops (ECLs) and intracellular loops (ICLs), and two nucleotide-binding domains (NBDs). ABC transporters export substrates through a series of conformational changes. Many ABC transporters shift between an inward-facing, drug-binding (high-affinity) conformation to an outward-facing, drug-releasing (loose-binding) conformation using ATP binding and hydrolysis. When the drug is released from the drug-binding pocket, it must be prevented from reentering through the transporter. The outward-facing or drug-releasing conformation results in only a 10-to-30-fold reduction in binding affinity, which is not sufficient to prevent some rebinding of the drugs if the extracellular concentration is high (Sauna and Ambudkar, 2000). For this reason, it was proposed that ABC transporters have a molecular gate or diode that prevents reflux (Gupta, et al., 2011). The first evidence for a molecular diode mechanism was the observation that a Ser 1368 Ala (S1368A) mutation in the yeast multidrug transporter Pdr5 exhibited significant drug reflux during transport (Mehla, et al., 2014). More recently, multiple cryo-electron microscopy structures of the ABCG2 mammalian multidrug transporter clearly showed the presence of a molecular gate between an inner and outer drug-binding pocket. Two prominent residues—Leu-554 and Leu-555—were identified as constituents of this gate (Manolaridis, et al., 2018). When the sequences of Pdr5 and ABCG2 are aligned, Ser-1368 is quite close to Leu-554 and Leu-555. The corresponding residues in Pdr5 are Val-1372 and Met-1373. Bioinformatics analysis of the Pdr subfamily (Lamping, et al., 2010) revealed highly conserved residues adjacent to Ser-1368, including Phe-1369, Gly-1371, Val-1372, and Met-1373. The corresponding residues to this S1368 adjacent motif in the amino-terminus of Pdr5, extending from Tyr-680 to Ilv-685, also exhibit conservation. The first goal of this study is to determine whether the conserved set of amino acids spanning residues (1369–1373) in the carboxyl-terminus motif of Pdr5 make up a molecular diode motif. The second goal is to investigate whether the molecular diode is composed of residues from both halves of this large, polytopic protein. Our results suggest that this conserved motif plays a variety of roles including signal transmission, protein folding, and molecular gating. Our results also suggest that Ile-685, Val-1372, and Met-1373 are the actual gate, where Ser-687, Ser-1368, and Phe-1369 provide structural support. Alanine substitutions in these residues exhibit a unique gating-defect phenotype. Characterization of mutants in these two motifs might lead to specific chemical inhibitors of these transporters during disease treatment., Biology, Biology, Degree Awarded: Ph.D. Biology. The Catholic University of America
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- Understanding and Presence: The Literary Achievement of the Early Modern Sermon
- In both their preached and printed forms, sermons were one of the dominant genres of the early modern period. While studies of early modern sermons tend to emphasize conflict by centering sermons’ occasional content, this dissertation approaches them primarily as literary texts. It explores how the sermons of Lancelot Andrewes, John Donne, Richard Sibbes and Henry King sought to move their auditories toward faith and in doing so worked to build a community. While Andrewes, Donne, Sibbes and King differed in style, theology and temperament, all four belonged to the “conforming center” of the Jacobean and Caroline English church. These four preachers offer a window into efforts within the conforming church to emphasize shared ground and, in the face of narrowing definitions of conformity on the one hand and of the true church on the other, to create a wider space within the national English church. In treating these sermons seriously as literary texts, this study offers close readings of selected individual sermons from each preacher. These readings join a growing number of studies of religious writing during this period that seek to examine how faith prompted literary efforts to bind rather than divide.These sermons, in language that rendered the abstractions of faith experiential, sought to evoke the presence of things not seen. This study argues that, in the sermons of these four preachers, the nature of that experience also shaped communal identity. The sense of the communal created by these sermons is one that explicitly seeks unity despite conflict through the creation of shared experience, rather than enforcing unity by drawing tighter boundaries. As such, these efforts represent attempts to articulate a unified experience of faith in the face of the increasing fragmentation of the church in England. This study traces, in Andrewes’ sacramental language, Donne’s performative recognition of experience, Sibbes’s loving mysticism, and King’s genreblurring, the ways some preachers of the conforming center sought to draw their auditories toward a participation in the sermon that was also participation in the community of faith. In distinctive ways, each preacher seeks to make apprehensible and immediate the remote things of faith., English literature, Andrewes, Community, Donne, Early Modern, Literature, Sermons, English Language and Literature, Degree Awarded: Ph.D. English Language and Literature. The Catholic University of America
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- Understanding the Role of RUNX1-TLE1 Interaction in the Pathogenesis of Familial Platelet Disorder with associated Myeloid Malignancy
- Megakaryopoiesis is the developmental process of megakaryocytes, which are the cells responsible for platelets production. This process is defective in patients with Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM). FPDMM patients are presented with decreased platelet count and/ or function. Importantly, 20-60% of FPDMM patients develop hematological malignancies later in their lives. FPDMM’s defective megakaryopoiesis is caused by a heterozygous germline mutation in the Runt-related Transcription Factor-1 (RUNX1). RUNX1 is essential during definitive hematopoiesis and megakaryopoiesis.FPDMM family was identified carrying a novel GC insertion in the RUNX1 gene, leading to a frameshift mutation (L472fsX). This caused a disruption of the VWRPY domain, a conserved binding site for the repressor protein, Transducin-like Enhancer of split-1 (TLE1). The significance of RUNX1 and TLE1 interaction in megakaryopoiesis and FPDMM pathogenesis has not been studied yet. To address those questions, we evaluated the functional defects of the RUNX1 L472fsX mutation in three different experimental models. In cell lines, FRET assay and Co-immunoprecipitation revealed loss of binding between RUNX1 L472fsX mutant and TLE1. In addition, transactivation assays against RUNX1’s target promoters, Csf1r and Hmga2, showed loss of TLE1’s repression effect on RUNX1 L472fsX mutant activity compared to wild type RUNX1. These preliminary data are consistent with the proposed regulatory role for RUNX1 and TLE1 interaction during hematopoiesis. Moreover, the findings support a gain of function mutation, which presents a new mechanism of RUNX1 action in FPDMM pathogenesis. To evaluate these defects in our FPDMM patients, we reprogrammed the patients’ cells to induced Pluripotent Stem Cells (iPSCs). Differentiating the patients’ iPSCs revealed a defect in their megakaryopoiesis similar to previous FPDMM studies. In addition, the patients’ iPSCs showed defects in Hematopoietic Stem Cells (HSCs) maturation and differentiation. This finding is first reported with this RUNX1 L472fsX mutation since no other FPDMM studies reported a similar phenotype. Significantly, these hematopoietic defects were rescued after correcting the RUNX1 L472fsX mutation in the patients’ iPSCs. Moreover, a mouse model missing the VWRPY domain expression was generated using CRISPR-Cas9 system. The homozygous mouse model showed minor defects in the peripheral blood, but not in the bone marrow. In conclusion, the data indicates the significance of RUNX1 and TLE1 interaction through the VWRPY domain to ensure efficient hematopoiesis and megakaryopoiesis, essentially in HSCs maturation and differentiation. More importantly, this RUNX1 L472fsX mutation presents a novel mechanism of FPDMM pathogenesis. It would give more insights into RUNX1’s function in hematopoiesis and how would FPDMM patients develop hematological malignancies., Cellular biology, FPDMM, iPSCs, Megakaryopoiesis, RUNX1, TLE1, VWRPY, Biology, Degree Awarded: Ph.D. Biology. The Catholic University of America
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- Thirteenth-Century English Religious Lyrics, Religious Women, and the Cistercian Imagination
- Degree awarded: Ph.D. Medieval and Byzantine Studies. The Catholic University of America, This dissertation can be viewed by CUA users only., Nearly all of the brief Middle English lyric poems that began to appear in manuscripts during the first half of the thirteenth century are religious in nature, and nearly all either concern the passion of Christ or are prayers to his mother, Mary. Very often the two motifs appear in tandem, in poems that place both speaker and audience at the foot of Christ's cross where Mary is engulfed in a sorrow that the reader is asked to experience empathetically. This dissertation argues that the lyrics grew out of a prose meditative genre, in particular a Cistercian meditative genre related to twelfth-century exegesis of the Song of Songs, that offered readers a series of visual tableaux of events in the life of Christ to experience imaginatively. The passion of Christ was a central focus of this sort of meditation. The English Cistercian abbot Aelred of Rievaulx's De institutione inclusarum, a treatise addressed to his anchoress-sister, offered a model of this genre that was widely copied and imitated, and some of the earliest English religious lyrics appear either as part of those prose meditative texts or as appendices thereto. Eventually both the prose texts and the lyrics became devotional reading for laypeople. This dissertation examines the literary relationship between the lyrics and the prose texts and some of the manuscripts where the earliest Middle English religious lyrics appear., Made available in DSpace on 2012-09-11T17:13:00Z (GMT). No. of bitstreams: 1 Allen_cua_0043A_10183display.pdf: 1722200 bytes, checksum: a6ac5c3174f99cdaed57a13b4270e82b (MD5)
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- Leadership Behaviors of the Catholic Elementary School Principal Which Support Novice Teachers
- Degree Awarded: Ph.D. Education. The Catholic University of America, The purpose of this study was to examine how the leadership behaviors of the Catholic elementary school principal shaped the culture of the school in support of novice teachers. When principals ensure that their schools are filled with good teachers, they help to raise the achievement level of the students. However, becoming a good teacher takes time and support. Novice teachers need support if they are to become effective practitioners. Through their leadership behaviors, Catholic elementary school principals can provide necessary supports for novice teachers. It is vital that Catholic elementary school principals understand how their leadership behaviors impact novice teachers. This research study employed a quantitative method which utilized a web-based self-administered survey. Data were obtained from 74 novice teachers in the nine Catholic Diocese of: the Archdiocese of Boston, the Archdiocese of Kansas City, the Archdiocese of St. Louis, the Archdiocese of Seattle, the Diocese of Austin, the Diocese of Des Moines, the Diocese of Fort Worth-South Bend, the Diocese of Jackson, and the Diocese of Phoenix. The survey instrument, The Catholic Elementary School Novice Teacher Survey, elicited demographic information, and provided data on the relationship focused and task focused leadership behaviors of the principal from the novice teacher's prospective. A Pearson product correlation coefficient and two-way analysis of variance were used for data analysis. The most important finding of this research study is that the leadership behaviors of the Catholic elementary school principal do shape the culture of the school in support of novice teachers. Both relationship focused leadership behaviors and task focused leadership behaviors were shown to positively impact the culture of the school in support of novice teachers. However, novice teachers' perceived relationship focused leadership behaviors of the principal to provide a higher level of support. It is important that principals understand their leadership behaviors in order to provide the necessary support for the development of the novice teachers in their school buildings.
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- The Impact of Dietary Iron Restriction on Sickling Kinetics and Hemolysis in A Transgenic Model of Sickle Cell Disease
- Sickle cell disease (SCD) is caused by polymerization of deoxy-hemoglobin-S (HbS) within the red blood cell (RBC). HbS polymerization is a concentration-dependent process. I hypothesized that dietary iron restriction would decrease the concentration of HbS in the RBC, resulting in delayed sickling, and decreased organ injury.To test this hypothesis, I studied male and female transgenic mice that express the human gamma, alpha and beta globin-S genes (SCD mice). To induce iron deficiency, mice were fed an iron-restricted diet (0-3 ppm iron) introduced at weaning. For comparison, parallel cohorts were fed an iron-sufficient diet (39 ppm iron) or an iron-abundant diet (821 ppm iron) also introduced at weaning. After 2, 4, or 6 months on the diets, mice were euthanized to collect blood and harvest tissues. Iron availability was determined by measuring plasma ferritin and hepcidin using ELISA kits. RBC and reticulocyte parameters were measured by an automated hematology analyzer. Hemoglobin composition was quantified by ion-exchange chromatography. Hb-O2 affinity was measured by sickling assay and HEMOX-Analyzer. RBC sickling was measured as the half-life of cells remaining round after exposure to 3% oxygen. Hemolysis was evaluated by measuring the absorbance of plasma heme, and the concentration of the heme and hemoglobin scavengers hemopexin and haptoglobin by ELISA. To examine tissues, animals were first perfused PBS. Organs were weighed to determine organ/body mass indices. Liver, spleen, and kidneys were preserved in formalin and embedded in paraffin for sectioning. Sections were stained with Prussian blue to determine iron deposition and stained with hematoxylin and eosin for histologic analysis by a veterinary pathologist. Histologic evidence of hepatic necrosis was reported as either present or absent.Dietary iron restriction caused depletion of iron stores as evidenced by lower plasma ferritin levels and suppression of the negative iron regulator hepcidin in both male and female SCD mice. RBC and reticulocyte parameters were unaffected by dietary iron restriction, though sex differences and age-related changes were observed. Fetal hemoglobin (HbF) levels were less than 1% and were unchanged by dietary iron restriction, though HbF levels were higher in females over time. Dietary iron restriction increased hemoglobin oxygen affinity in the first cohort, but this finding was not confirmed in a second cohort. The half-life of round cells under conditions of 3% oxygen was increased in SCD mice after 4 or 6 months of dietary iron restriction, but not after 2 months. The plasma heme concentration was decreased by the dietary iron-restriction at 4 and 6 months in the first cohort but was unchanged in the second cohort. Liver mass indices were decreased in mice fed the iron-restricted diet, and dietary iron restriction reduced the incidence of hepatic necrosis in the females by half.Dietary iron restriction was effective at depleting iron stores and suppressing the negative regulator of iron uptake, hepcidin. Nevertheless, the concentration of hemoglobin S, and many other RBC parameters, remained constant despite a clear impact of diet on iron stores and regulation. The physiologic effects of iron restriction showed important sex-specific differences: male SCD mice developed increased hemoglobin oxygen affinity and a longer round cell half-life after six months of iron restriction; whereas female SCD mice were protected against hepatic necrosis without similar improvements in sickling. My work demonstrates that iron restriction can improve sickling through changes in hemoglobin oxygen affinity, rather than through changes in hemoglobin-S concentration, as hypothesized. Furthermore, I have demonstrated for the first time that iron restriction is protective against hepatic necrosis, without changes in ex vivo sickling. This raises the possibly that protection is conferred instead through sex-specific increases in hepatic iron recycling. Together, the results in this thesis show that iron restriction can modulate the severity of sickle cell disease through both erythrocytic and non-erythrocytic pathways – a key conclusion that will inform clinical studies of iron restriction in patients with sickle cell disease., Health sciences, Hematology, Iron Deficiency Anemia, Red Blood Cell, Sickle Cell Disease, Biology, Degree Awarded: Ph.D. Biology. The Catholic University of America
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- A Linguistic Analysis of the Garshuni Of MS. Jerusalem, Monastery of St. Mark 199 (1732-1733)
- This work aims to provide a linguistic analysis of the Garshuni language attested in MS. Jerusalem, Monastery of St. Mark 199 (1732-1733). Garshuni refers to the Arabic language written in Syriac script starting as early as 12th century to modern time. Garshuni language is part of the larger term “Middle Arabic” which has its own issues in term of history and linguistic aspects. Focusing on the Syntactical issues in this manuscript, the study looked at what is seen as deviation from Classical Arabic by Arab grammarians as part of the linguistic development. Despite the work has been done on Middle Arabic to date, no linguistic analysis has been conducted on Garshuni. We lack scholarly studies of individual Garshuni text that could help us had better understand how this variety of Middle Arabic fits into the history of Arabic language more broadly. My method in this work is to analyze over 120 pages of the manuscript and more than 430 cases by comparing them with Classical Arabic, other Semitic languages, and Modern Arabic dialects. The findings provide some different answers to the Middle Arabic deviations, which normally accused by pseudo-corrections. Although pseudo-correction was the main reason of the deviation in some cases, there is a great amount of cases has equivalent usage in Classical Arabic or Modern Arabic dialects or sometimes both. Thus, this work, with the new findings, adds to the field of Arabic language history another approach by analyzing more Middle Arabic texts instead of look at them as a lower level of Arabic., Linguistics, Middle Eastern studies, Language, Arabic, Christian, Garshuni, Semitic and Egyptian Languages and Literatures, Degree Awarded: Ph.D. Semitic and Egyptian Languages and Literatures. The Catholic University of America
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- Bruton Tyrosine Kinase Inhibitors in Chronic Lymphocytic Leukemia Patients: Pharmacodynamic Analysis and Mechanisms of Resistance
- Chronic lymphocytic leukemia (CLL) is a malignancy of mature B cells that depend on host factors in their tissue microenvironment for growth and survival, primarily mediated by B-cell receptor (BCR) signaling. Targeting Bruton's Tyrosine Kinase (BTKis) downstream of the BCR has emerged as a successful strategy for CLL treatment. These drugs inhibit BTK by irreversible covalent binding to cysteine 481 in the BTK active site. A measure of on-target effects for these agents is the fraction of drug-bound BTK (occupancy), typically exceeding 80% for all covalent BTKis in clinical use. How levels of BTK occupancy correlate to inhibition of intracellular signaling and anti-tumor effects were not defined. Additionally, BTKis disrupt the interaction of CLL cells with their microenvironment, displacing leukemic cells from the lymphoid organs and causing transient lymphocytosis in CLL patients. CD49d, the α chain of the α4β1 integrin, plays an important role in the interaction of CLL cells with their microenvironment and is one of the main adverse prognostic markers in CLL. However, the underlying biological mechanisms of how CD49d expression impacts responses to BTKis remain elusive. In this study, to better understand the mechanisms of action of BTKis, we investigated the relationship between BTK occupancy and inhibition of downstream signaling. Further, we investigated the role of CD49d in response to BTKis in CLL patients treated with acalabrutinib, one of the approved, highly selective covalent BTKi. Our study design included randomization of CLL patients to once or twice daily dosing of acalabrutinib, and dose interruptions for 48 hours during the first week. We evaluated ex vivo BTK occupancy and readouts of BCR signaling, including target gene expression and protein-based readouts, sequentially between 4 and 48 hours from the last dose. Four hours from the last dose, we observed complete saturation of BTK and significant inhibition of BTK-dependent signaling in both dosing regimens. At trough 12 and 24 hours after the last dose, BTK occupancy was higher with twice-daily (median 95%) than with once-daily dosing (median 87%). We observed superior inhibition of target gene expression with twice-daily dosing, suggesting that higher BTK occupancy translates into more potent inhibition of oncogenic signaling. We next correlated the level of free BTK with endogenous and exogenously activated BCR. During the dosing interruption period, responses to exogenous BCR activation were proportional to the levels of free BTK generated by de novo synthesis. Together, higher BTK target occupancy was achieved with twice-daily dosing over once-daily dosing, resulting in deeper and more sustained inhibition of BCR signaling. To elucidate the role of CD49d in response to BTKis, CLL patients were classified into CD49d+ and CD49d− based on CD49d expression according to the clinically validated 30% cutoff. We first analyzed CD49d expression and activation changes during acalabrutinib treatment using flow cytometry. In addition, we evaluated the clinical responses during therapy and performed transcriptomic analyses comparing gene expression changes between the CD49d groups. Clinically, CD49d+ and patients with bimodal CD49d expression, i.e. patients with concurrent CD49d positive and negative CLL cells irrespective of the size of the CD49d+ population, had a significantly higher risk of progression on acalabrutinib than CD49d− patients. CD49d+ patients showed less treatment-induced lymphocytosis than CD49d− patients. In bimodal patients, the proportion of CD49d+ CLL cells decreased on treatment, suggesting that CD49d+ cells are better able to migrate and re-enter lymphoid tissues. While acalabrutinib treatment inhibited CD49d activation, in vitro stimulation of BCR and CXCR4 signaling restored CD49d activation. Furthermore, as revealed by gene set enrichment analysis, we observed higher levels of cellular activation, proliferation, adhesion, and survival in CD49d+ CLL cells. These results suggest that CD49d may contribute to the development of BTKi resistance in CLL by enhancing cell survival through adhesion to the tumor microenvironment. Overall, our study supports dosing strategies that maximize BTK occupancy for more therapeutic efficiency. Furthermore, CD49d expression increases the risk for the development of BTKi resistance and serves as an adverse prognostic marker. Further investigation how targeting CD49d could improve BTKi therapy is warranted. , Oncology, Immunology, Pharmacology, Bruton tyrosine kinase inhibitor, CD49d, Chronic lymphocytic leukemia (CLL), Targeted therapy, Biology, Degree Awarded: Ph.D. Biology. The Catholic University of America