Biology Dissertations

Characterization of high-persister strains in Mycobacterium tuberculosis

Heather Leah Torrey — Fri, 14 Jun 2013 08:55:47 PDT

Bacterial persister cells are phenotypic variants of the wild-type that are highly tolerant to bactericidal antibiotics. It is thought that persister cells may be responsible for recurrent bacterial infections due to the inability of antibiotics to eradicate them. We suggest that the recalcitrance of tuberculosis to antibiotic therapy may be due to the presence of persister cells. To identify genes responsible for persister formation in Mycobacterium tuberculosis, we have generated high-persister mutants in vitro that produce between 10- and 1,000-fold more persisters than the parental strain. Through whole-genome sequencing and transcriptome analysis, we have identified several candidate genes, some of...

Structure function analysis of Escherichia coli pol IV

Ryan W. Benson — Fri, 07 Jun 2013 08:54:26 PDT

Translesion synthesis (TLS) DNA polymerases are low fidelity DNA polymerases responsible for inserting a deoxynucleotide opposite to and extending from replication-blocking DNA lesions that have evaded high fidelity DNA repair pathways. E. coli DinB (DNA Pol IV) is a TLS polymerase of great interest because of its conservation throughout all domains of life and its relatively high intracellular basal level when compared to other DNA polymerases. Alternative activities of DinB other than its TLS ability include: roles in recombination, transcription, and a DNA replication "checkpoint." Using a structure/function strategy, we sought to better understand these activities by utilizing dinB alleles...

Bistability in the protozoan parasite Giardia lamblia

Alimatu Sulemana — Wed, 06 Mar 2013 10:23:49 PST

Giardia lamblia is an important protozoan parasite, the transmission of which from one host to the other, and survival outside the host are dependent on differentiation of vegetative trophozoites into infectious cysts (encystment). Like many protozoa, Giardia trophozoites differentiate into cysts in response to nutrient deprivation or other unfavorable environmental changes. Giardia, a lipid auxotroph, encysts when bile sequesters lipids that they require (Jarroll et al., 1981, Luján et al., 1996b). During encystment, Giardia trophozoites elicit a unique pattern of gene expression resulting in the synthesis and transport of cyst wall components ultimately resulting in the formation of a protective...

Response of the larval zebrafish to spinal cord injury: labeled lesions, two-photon axotomy and recovery of visuomotor behaviors

Sucharita Saha — Fri, 11 Jan 2013 08:02:05 PST

Spinal cord injuries can result in near total loss of function below the spinal-level of damage, due to the interruption of descending neural commands. A number of approaches are being tested to enhance axonal regeneration and assess the recovery of spinal cord function although, to date, no effective regeneration has been observed following complete spinal transection. Given the limited functional recovery seen in a variety of studies, and a lack of understanding of underlying molecular mechanisms, a system that enables faster and more precise analyses is needed. We attempt here to lay a foundation for such studies in larval zebrafish....

Filamin is required for F-actin organization and calcium signaling in C. elegans

Ismar Kovacevic — Fri, 04 Jan 2013 10:24:07 PST

During organism development cells sense, integrate, and respond to a variety of cues or signals. Mechanical properties of cells, and the matrix that surrounds them, contribute critical signals that direct cell migration and differentiation. In this study we sought to establish a simple model system to study mechanotransduction in an intact organism at the cellular level. The C. elegans spermatheca is a simple organ, consisting of a layer of myoepithelial cells enclosed by a basement membrane. Oocytes are ovulated into the spermatheca, where they are fertilized, and then propelled into the uterus by constriction of the spermatheca. Phenotypic analysis of...

Positive cross-talk between ecdysone and insulin signaling in insect growth

Srikanth Subramanian — Wed, 28 Nov 2012 06:40:25 PST

Wing imaginal discs are masses of undifferentiated cells that give rise to the forewings and hindwings of adult insects. In this dissertation, I examined the roles of insulin and ecdysone, a steroid molting hormone, in the regulation of imaginal wing disc growth in the tobacco hornworm, Manduca sexta. Previous studies on a different lepidopteran insect had suggested a requisite role for both hormones in disc growth providing a means to coordinate nutritional regulation of growth with metamorphic development. I studied the short-term and long-term stimulation of signaling events in the insulin pathway, and in ecdysone receptor content, in order to...

The conserved gene CACN-1 controls cell migration and germline differentiation in C. Elegans

Hiba Tannoury — Wed, 24 Oct 2012 10:24:40 PDT

Cell migration is fundamental to the development and physiological maintenance of organisms. Studying how cell migration is regulated is critical to the understanding of several developmental disorders, and it may help develop novel therapeutic approaches to disease conditions caused by erroneous cell migration, such as metastatic tumors. In the C. elegans nematode, the two specialized distal tip cells (DTCs) migrate long distances during nematode development, and therefore provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC...

Life cycle, novel proteins and structural organization of the synechococcus cyanophage Syn5

Desislava Andreeva Raytcheva — Wed, 06 Jun 2012 05:33:48 PDT

Syn5 is a marine cyanophage, which is propagated under laboratory conditions on the marine cyanobacterial strain Synechococcus sp. WH8109. Its genome has high homology with enteric bacteriophage T7 but it also contains several genes encoding protein sequences with no matches to phage proteins in the NCBI database. Cryo-EM images of Syn5 revealed a novel structure, a so-called horn, on the vertex opposite the tail.

Synchronized, exponentially growing host cultures were cultivated and the one-step growth curve of Syn5 was performed. Syn5 exhibited a short life cycle with an eclipse period of about 43 min and a latent phase of about...

Protistan species and their diversity from a molecular perspective

Angela Schena — Mon, 07 May 2012 10:20:46 PDT

Traditionally protists have been described based on their morphology using what is often referred to as alpha taxonomy approaches. However, today it is all but certain that these approaches have not revealed the real scale of protistan diversity. The two main reasons are the current uncultivability of most of microbial eukaryotes, which often makes direct observations impossible, and the lack of a comprehensive concept of species. Today, the molecular based (beta) taxonomy of comparisons of DNA sequences is increasingly used to bypass the limitations of the alpha taxonomy. The challenge is that the two approaches are typically used separately, and...

Contributions of zebrafish descending motor control neurons to visually-guided locomotor behaviors

Kristen Eileen Severi — Wed, 21 Mar 2012 05:50:57 PDT

The descending motor control neurons of the larval zebrafish, including the reticulospinal cells, vestibulospinal cells, and the nucleus of the medial longitudinal fasciculus (nMLF), are the primary pathway by which locomotor information is transmitted to the spinal cord. They number approximately three hundred, in many cases are individually identifiable, and act as the sole pathway of information. This makes them amenable to precise study and characterization and allows us to gain some insight into the control of locomotion. We characterize the activation patterns of the array of descending neurons during behavioral maneuvers such as slow swimming and routine turns in...

The influence of environmental stresses on Escherichia coli tolerance to antimicrobials

Yanxia Wu — Fri, 16 Mar 2012 09:45:22 PDT

Persisters are bacterial cells that survive antibiotic treatment without acquiring resistanceconferring mutations. Upon antibiotic removal, they form a population identical to the parental one, complicating treatment of infectious diseases. Persisters are multidrug-tolerant and form in response to stresses. Persisters can form in response to fluoroquinolone (FQ) treatment through the induction of the SOS response. FQ treatment produces DNA double strand breaks (DSBs), which induce the SOS response. TisB, part of a toxin-antitoxin (TA) module, is induced by the SOS response and causes persister formation by decreasing the proton motive force and ATP levels. Some persisters also form prior to antibiotic...

The anti-hypoxia-adenosinergic approach to the immunotherapy of cancer

Stephen Matthew Hatfield — Wed, 07 Mar 2012 09:31:43 PST

The suppression of tumor-reactive T- and NK cells in the tumor microenvironment (TME) involves several functionally distinct mechanisms that include the modulation of immunological negative regulators, suppressor cells, and the secretion of soluble anti-inflammatory factors that allow tumors to escape immunosurveillance. In addition to these mainstream mechanisms of immunosuppression in the TME, Sitkovsky and co-authors have suggested that it is the tumor hypoxia-driven accumulation of extracellular adenosine and subsequent signaling by engagement of the cAMP-elevating A2 adenosine receptors on tumor-reactive effector T-7 or NK cells8 that is instrumental in aiding the process of tumor evasion. Here, we show that the...

Protistan microbial observatory in the Cariaco Basin: an examination of species richness, habitat specialization, and biogeography

William Orsi — Wed, 15 Feb 2012 06:42:21 PST

The largest 18S rRNA Sanger sequence dataset from an oceanographic regime is combined with pyrosequencing to interrogate protistan diversity in the Cariaco Basin. A multiple PCR primer approach was applied to a total of 16 samples from 3 locations across the Basin and 4 depths along the basin's geochemical gradients. Phylogenetic analyses identify new clades at multiple levels of taxonomic hierarchy, revealing increased ecological and geographic distributions. Several clades were detected only in anoxic samples, suggestive of habitat specialization. This is supported by multivariate analyses and parametric richness estimations showing a division between communities present in different geochemical layers of...

Modulating angiogenesis through the Th1 and Th2 immune switch

Alexander Stephan Schering — Thu, 01 Dec 2011 07:51:27 PST

Angiogenesis and inflammation are inherently interconnected processes. Defects in the natural homeostasis of those processes leads to the pathogenesis of several major diseases, such as diabetic retinopathy (DR), age-related macular degeneration (AMD), tumorigenesis and asthma. A major commonality between those diseases is that the increased formation of abnormal vasculature constitutes a rate-limiting factor. Thus, characterizing and understanding the unique functional properties of this abnormal vasculature in vivo is crucial for the development of successful therapies.

In this dissertation we set out to establish an in vivo , microparticle based, molecular imaging approach for the purpose of studying adhesion molecules in...

Ontogenetic changes in the sequencing, kinematics and sensory control of zebrafish feeding episodes

Rebecca Elizabeth Westphal — Tue, 29 Nov 2011 08:03:05 PST

Successful feeding is a ubiquitous requirement of all animals. In many larval fishes feeding involves orienting towards and capturing motile prey items. Early larval zebrafish prey capture has previously been described. Here, we identify changes to this behavior throughout ontogeny. The individual tracking maneuvers present in early larval fish seem to be fused with the final strike to form a single, fluid maneuver in juvenile and adult zebrafish. Coincident with this behavioral streamlining, drastic changes in peak velocity, tail beat frequency and other kinematic variables are occurring. Teleost feeding requires finely tuned sensory systems that are capable of guiding the...

The scout model: a new viewpoint on microbial survival strategies

Sandra M. Buerger — Fri, 04 Mar 2011 06:59:19 PST

It has been frequently observed that microorganisms from different sources exist in a non-growing state. We suggest that this state represents a population level strategy utilized by non-sporeforming bacteria to maximize survival under unfavorable conditions. Individual members of the population enter a state of reversible dormancy from which they exit in a seemingly random fashion due to noise in gene networks. We term these stochastically awakened cells scouts, and further hypothesize that scouts may produce signaling compounds that cue the remaining dormant population to initiate growth (Epstein 2009a). To test this model, we employed single cell incubations from a mixed...

Regulation of circadian rhythm through SCN and non-SCN factors

Harry Pantazopoulos — Fri, 04 Mar 2011 06:29:01 PST

It is well known that the suprachiasmatic nucleus (SCN) is the master clock in mammals, however, how the SCN signals to other brain areas to regulate circadian rhythms is not yet known. The first two chapters in this thesis explore potential SCN output signals that may serve to entrain slave oscillators in other brain regions and other organs. Infusion of agonists or antagonists for the VIP receptor, as well as prokineticin 2, into the third ventricle of hamsters identified the role of VIP signaling in regulating light effects on the SCN in comparison to the role of prokineticin 2 as...

Genes responsible for antibiotic tolerance in escherichia coli

Sonja Hansen — Wed, 22 Dec 2010 12:12:39 PST

Bacterial populations stochastically produce a small number of non-growing or dormant persister cells that are tolerant to antibiotics. Persisters are phenotypic variants that are genetically identical to the susceptible cells within a clonal population. Persisters contribute to the antibiotic recalcitrance of biofilm infections. A number of recent studies point to the involvement of toxin/antitoxin (TA) systems in persister formation. A strain with two point mutations in hipA, the toxin of the hipBA TA system, produced 10,000 times more persisters than the wild type. Similarly, overexpression of HipA caused a sharp increase in the persister fraction. However, deletion of hipA did...

SOS response and the mechanism of adaptive tolerance in Escherichia coli

Tobias Dörr — Fri, 10 Dec 2010 09:15:14 PST

Bacteria produce persisters, a small subpopulation of cells that neither grow nor die in the presence of antibiotics. Persisters are tolerant against exposure to multiple antibiotics and they likely contribute to the relapse of bacterial infections after antibiotic therapy. The mechanism of persister formation is unknown, although several studies have pointed towards redundancy in persister formation mechanisms and the possible involvement of chromosomal toxin-antitoxin modules. While studying the genetic requirements for Escherichia coli persister survival after exposure to the DNA damaging antibiotic ciprofloxacin, we found that persister formation was an adaptive response to the antibiotic. Survivors to ciprofloxacin exhibited low...

Breeding strategies and the reproductive ecology of nasutitermes corniger

Tamara R. Hartke — Thu, 23 Sep 2010 07:57:11 PDT

The phylogenetically-derived Neotropical termite Nasutitermes corniger is known for its facultatively polygamous mating strategy. It has been hypothesized that these associations of multiple unrelated queens and kings come about through pleometrosis, a process of group colony foundation well-studied in the social Hymenoptera, but virtually unexplored in the termites. To better understand the dynamics of colony foundation, and in particular the costs and benefits of pleometrosis compared to monogamy, experimental incipient colonies were established in conjunction with field observations and choice trials under semi-natural conditions. The results presented in this dissertation suggest no clear benefit of pleometrotic colonies over those established...