<p> <i>Wolbachia</i> are gram-negative, obligate, intracellular bacteria infecting a majority of insect species and filarial nematodes. In both insects and nematodes <i>Wolbachia</i> are primarily transmitted through the female germ line. <i>Wolbachia</i> carried by filarial nematodes are the cause of the neglected diseases African river blindness and lymphatic filariasis afflicting millions worldwide. In order to combat these diseases, we created a <i>Wolbachia</i>-infected Drosophila cell line that enabled high throughput screening for novel potent anti-<i> Wolbachia</i> compounds. Of the 36,231 compounds screened in house, 8 compounds dramatically reduced <i>Wolbachia</i> titer both in the cell and nematode based screen. Significantly, we discovered that the albendazole metabolite, albendazole sulfone, reduces <i>Wolbachia</i> titer in Drosophila melanogaster and the filarial nematode <i>Brugia malayi </i> perhaps by directly targeting <i>Wolbachia</i> FtsZ. Using the <i>Wolbachia</i>-infected cell line, we discovered that in addition to vertical germ line transmission, <i>Wolbachia</i> are efficiently transmitted horizontally via cell-to-cell transmission. We show that horizontal transfer is independent of cell-to-cell contact, can efficiently take place within hours, and uses both host cell phagocytic and clathrin/dynamin-dependent endocytic machinery. Modifications to our high-throughput screen in combination with genome-wide RNA interference (RNAi) identified host factors that influence <i> Wolbachia</i> titer. When these host factors were tested in <i> Drosophila melanogaster in vivo</i> we found that maintenance of <i>Wolbachia</i> titer relies on an intact host Endoplasmic Reticulum (ER) associated degradation (ERAD) system. These data, in combination with electron microscopy studies, demonstrated that <i>Wolbachia</i> is intimately associated with the host ER and suggested a previously unsuspected mechanism for the potent ability of <i>Wolbachia</i> to prevent RNA virus replication. To examine the impact of nutritional on <i>Wolbachia </i> titer, Drosophila were fed sucrose- and yeast-enriched diets. These conditions resulted in increased and decreased <i>Wolbachia</i> titer in Drosophila oogenesis, respectively, and that somatic TOR and insulin signaling mediate the response of the yeast-enriched diet on <i>Wolbachia </i>. Taken together, these studies provide initial insights into the molecular and cellular interactions between <i>Wolbachia</i> and its insect and nematode hosts.</p><p>

<p> Progression through the mammalian cell cycle is a tightly regulated process that allows cells to replicate their genomes and divide properly. In growth factor-deprived conditions or in response to stress, the cell will exit the cell cycle either reversibly through quiescence, or permanently via senescence. Studies have shown that the SIN3 family of proteins plays a crucial role in these cell cycle exit processes. SIN3 proteins are highly conserved, and exist in mammals as two family members: SIN3A and SIN3B, which function as flexible scaffolding proteins to assemble co-repressor complexes. Our laboratory has recently implicated SIN3B as a critical mediator of each of these cell cycle exit processes. However, its mechanism of action and the consequences of its disruption pertaining to cancer progression have not been comprehensively elucidated. Here we demonstrate that SIN3B is required for the induction of senescence in a mouse model of prostate cancer, and thus prevents the progression to aggressive and invasive carcinoma. In addition, through interaction analysis, we uncovered a novel and robust association between SIN3B and the DREAM complex. The DREAM complex, comprised of p107/p130, E2F4/5, DP1 and the MuvB core complex, is responsible for the repression of hundreds of cell cycle-related transcripts during quiescence. We determined that the deletion of <i>SIN3B</i> resulted in the derepression of DREAM target genes during quiescence, but was not sufficient to allow quiescent cells to resume proliferation. However, the ectopic expression of APC/C^CDH1 inhibitor EMI1 was sufficient for <i>SIN3B</i> deleted cells, but not wild-type cells, to reenter the cell cycle. These studies demonstrate a critical role for SIN3B in the senescence and quiescence programs, and provide important mechanistic insight into the molecular pathways that exquisitely regulate cell cycle exit.</p><p>

<p> Present day genomic technologies are evolving at an unprecedented rate, allowing interrogation of cellular activities with increasing breadth and depth. However, we know very little about how the genome functions and what the identified genes do. The lack of functional annotations of genes greatly limits the post-analytical interpretation of new high throughput genomic datasets. For plant biologists, the problem is much severe. Less than 50% of all the identified genes in the model plant <i>Arabidopsis thaliana,</i> and only about 20% of all genes in the crop model <i>Oryza sativa</i> have some aspects of their functions assigned. Therefore, there is an urgent need to develop innovative methods to predict and expand on the currently available functional annotations of plant genes. With open-access catching the &lsquo;pulse&rsquo; of modern day molecular research, an integration of the copious amount of transcriptome datasets allows rapid prediction of gene functions in specific biological contexts, which provide added evidence over traditional homology-based functional inference. The main goal of this dissertation was to develop data analysis strategies and tools broadly applicable in systems biology research. </p><p> Two user friendly interactive web applications are presented: The Rice Regulatory Network (RRN) captures an abiotic-stress conditioned gene regulatory network designed to facilitate the identification of transcription factor targets during induction of various environmental stresses. The <i>Arabidopsis </i> Seed Active Network (SANe) is a transcriptional regulatory network that encapsulates various aspects of seed formation, including embryogenesis, endosperm development and seed-coat formation. Further, an edge-set enrichment analysis algorithm is proposed that uses network density as a parameter to estimate the gain or loss in correlation of pathways between two conditionally independent coexpression networks.</p><p>

<p> Genome instability is a hallmark of human cancers. Patients with Bloom&rsquo;s syndrome, a rare chromosome breakage syndrome caused by inactivation of the RecQ helicase BLM, result in phenotypes associated with accelerated aging and develop cancer at a very young age. Patients with Bloom&rsquo;s syndrome exhibit hyper-recombination, but the role of BLM and increased genomic instability is not fully characterized. Sgs1, the only member of the RecQ family of DNA helicases in <i>Saccharomyces cerevisiae,</i> is known to act both in early and late stages of homology-dependent repair of DNA damage. Exo1, a 5'&ndash;3' exonuclease, first discovered to play a role in mismatch repair has been shown to participate in parallel to Sgs1 in processing the ends of DNA double-strand breaks, an early step of homology-mediated repair. Here we have characterized the genetic interaction of <i>SGS1</i> and <i> EXO1</i> with other repair factors in homology-mediated repair as well as DNA damage checkpoints, and characterize the role of post-translational modifications, and protein-protein interactions in regulating their function in response to DNA damage. In <i>S. cerevisiae</i> cells lacking Sgs1, spontaneous translocations arise by homologous recombination in small regions of homology between three non-allelic, but related sequences in the genes <i>CAN1, LYP1,</i> and <i>ALP1.</i> We have found that these translocation events are inhibited if cells lack Mec1/ATR kinase while Tel1/ATM acts as a suppressor, and that they are dependent on Rad59, a protein known to function as one of two sub-pathways of Rad52 homology-directed repair.</p><p> Through a candidate screen of other DNA metabolic factors, we identified Exo1 as a strong suppressor of chromosomal rearrangements in the <i> sgs1&Delta;</i> mutant. The Exo1 enzymatic domain is located in the N-terminus while the C-terminus harbors mismatch repair protein binding sites as well as phosphorylation sites known to modulate its enzymatic function at uncapped telomeres. We have determined that the C-terminus is dispensable for Exo1&rsquo;s roles in resistance to DNA-damaging agents and suppressing mutations and chromosomal rearrangements. Exo1 has been identified as a component of the error-free DNA damage tolerance pathway of template switching. Exo1 promotes template switching by extending the single strand gap behind stalled replication forks. Here, we show that the dysregulation of the phosphorylation of the C-terminus of Exo1 is detrimental in cells under replication stress whereas loss of Exo1 suppresses under the same conditions, suggesting that Exo1 function is tightly regulated by both phosphorylation and dephosphorylation and is important in properly modulating the DNA damage response at stalled forks.</p><p> It has previously been shown that the strand exchange factor Rad51 binds to the C-terminus of Sgs1 although the significance of this physical interaction has yet to be determined. To elucidate the function of the physical interaction of Sgs1 and Rad51, we have generated a separation of function allele of <i> SGS1</i> with a single amino acid change <i>(sgs1-FD)</i> that ablates the physical interaction with Rad51. Alone, the loss of the interaction of Sgs1 and Rad51 in our <i>sgs1-FD</i> mutant did not cause any of the defects in response to DNA damaging agents or genome rearrangements that are observed in the <i>sgs1</i> deletion mutant. However, when we assessed the <i>sgs1-FD</i> mutant in combination with the loss of Sae2, Mre11, Exo1, Srs2, Rrm3, and Pol32 we observed genetic interactions that distinguish the <i>sgs1-FD</i> mutant from the <i>sgs1 </i> deletion mutant. Negative and positive genetic interactions with <i> SAE2, MRE11, EXO1, SRS2, RRM3,</i> and <i>POL32</i> suggest the role of the physical interaction of Sgs1 and Rad51 is in promoting homology-mediated repair possibly by competing with single-strand binding protein RPA for single-stranded DNA to promote Rad51 filament formation.</p><p> Together, these studies characterize additional roles for domains of Sgs1 and Exo1 that are not entirely understood as well as their roles in combination with DNA damage checkpoints, and repair pathways that are necessary for maintaining genome stability.</p><p>

<p> This study examined various hybrid peptides that possess both collagen [(POG)_n] and cell penetrating peptides (CPP) [(RRG)_n or R_n] sequences. The hybrid peptides were able to fold into triple helical conformation when the surrounding temperature was lower than their transition temperature (T_m) which resulted in cellular internalization. The peptide that lacked collagen [(POG)_n] domain failed to penetrate the cell. The hybrid peptide under study, FL7V1, was shown to have the ideal T_m (17.3&deg;C) for the potential purpose as a drug carrier. <i>In vitro</i> study of FL6V1 with temperature gradient showed cellular internalization at low temperatures (10&deg;C-20&deg;C) while no uptake was achieved at high temperatures (24&deg;C-32&deg;C). <i> In vivo</i> study of FL7V1 with <i>P. leidyi</i> corresponded with the results of <i>in vitro</i> study at constant and gradient temperature.</p>

Fluorescent ratiometric imaging of Lilium longiflorum pollen tubes loaded with the Ca$\sp{2+}$ indicator fura-2 dextran has revealed a tip-focused gradient of free intracellular calcium ions in actively growing pollen tubes which extends from above 3.0 $\mu$M at the apex to a uniform basal level of $\sim$0.2 $\mu$M within 20 $\mu$m from the tip. Application of the Ca$\sp{2+}$ specific vibrating electrode revealed a tip-directed extracellular Ca$\sp{2+}$ influx between 1.4 and 14 pmol cm$\sp{-2}$ sec$\sp{-1}$. The relationship between these phenomena and their role in tube growth was examined using different 1,2-bis (o-aminophenoxy)ethane $N,N,N\sp1,N\sp1$-tetraacetic acid (BAPTA)-type buffers, and hypertonic media. Injection of active BAPTA-type buffers or application of elevated sucrose levels reversibly inhibited growth, destroyed tip zonation of organelles, and modified normal patterns of cytoplasmic streaming. Simultaneously, these treatments dissipated both the intracellular tip-focused gradient and the extracellular Ca$\sp{2+}$ flux providing evidence that growing pollen tubes have open Ca$\sp{2+}$ channels in their tip which become inactivated in nongrowing tubes. The elevated sucrose studies support the view that stretching of the apical plasma membrane contributes to the maintenance of the Ca$\sp{2+}$ signal. The presence and localization of actin and myosin have been examined in Lilium longiflorum and Nicotiana alata pollen tubes in order to determine their roles in pollen tube growth. Immunoblot analysis of pollen tube extracts with antibodies to actin, myosins IA and IB, myosin II, and myosin V reveals the presence of these contractile proteins. Immunofluorescence microscopy reaffirmed longitudinal actin localization. Myosin I was localized to the plasma membrane, larger organelles, the surface of the generative cell and the vegetative nucleus. Myosin V and Myosin II were found in the vegetative cytoplasm in a punctate fashion representing smaller and larger organelles, respectively. These results suggest the presence of three classes of myosins in pollen and lead to the following hypothesis: Myosin I may move the generative cell and vegetative nucleus unidirectionally toward the tip, while myosin V moves the smaller organelles and myosins I and II move the larger organelles (bidirectionally).

As an extension of the previous work on the pH-dependent reorganization of artificial lipid vesicles by poly(2-ethylacrylic acid) (PEAA), the effect of PEAA on living cells are examined by utilizing the acidification process of the epidermal growth factor (EGF) endocytic pathway in A431 cells. Fluoresceinated EGF (f-EGF) was used in conjunction with two-wavelength frame transfer fluorescence imaging techniques to measure the pH's of endocytic vesicles of individual cells. Preliminary results, including feasibility study and PEAA toxicity to A431 cells, are presented in chapter 3. Two projects branched out of this initial study; one involves the recording of PEAA channel activity using single-channel recording techniques (chapter 2), and another the measurement of binding kinetics of f-EGF to A431 cells at the single cell level (chapter 1). The synthetic anionic polyelectrolyte PEAA has been shown to reorganize lipid vesicles under acidic conditions. Single-channel recording techniques are used to further characterize the interactions between PEAA and artificial phospholipid bilayers. PEAA is shown to induce pore formation in bilayers at low (PEAA) ($<$ 20 $\mu$g/ml) in a pH-dependent manner. Discrete events resembling those characteristic of biological channels are observed. PEAA channels are found to be cation-selective, as demonstrated by comparing the relative Na$\sp+$ and Cl$\sp-$ permeabilities of the channels, but the Na$\sp+$/Cl$\sp-$ permeability ratio spans a wide range, indicating the existence of channels of different ion selectivities. Point amplitude histogram analysis of one type of channel event reveals at least four separate single-channel conductance states. The interpretation of ligand-receptor equilibrium binding data collected from a population of cells is often ambiguous. A method using quantitative fluorescence microscopy to measure EGF/receptor binding kinetics at the single cell level is developed. The binding of f-EGF to single cells is recorded over time and fitted to models with photobleaching correction and either one monovalent binding site or two independent monovalent binding sites. The results disprove the one-site model; therefore, the reported EGF receptor affinity heterogeneity exists at the single cell level.

The neuromuscular junction is the site where signals are transmitted from a nerve to a target muscle fiber. The mechanisms responsible for the maintenance of motor nerve terminals at synaptic sites are not understood. Here, I investigated the role of target-muscle fibers in the maintenance of frog motor nerve terminals. Cutaneous pectoris muscle fibers were selectively removed and prevented from regenerating while leaving the motor innervation intact. The role of target muscle fibers in nerve terminal structure and function was examined. First, the maintenance of presynaptic activity in the absence of target was assayed with the activity-dependent dye FM1-43. I found that target-deprived nerve terminals maintain their presynaptic function of synaptic vesicle recycling for up to 5 months of target deprivation. These results indicated that the molecular machinery required for vesicular release is maintained in a functional state for long periods of target deprivation. Second, I quantified the stability of target-deprived nerve terminals using in vivo repeated imaging. I found that most target-deprived nerve terminals were remarkably well maintained for several months after muscle fiber removal. These data indicate that the cues that confer stability to frog motor nerve terminals reside outside the muscle fibers such as in the synaptic basal lamina or terminal Schwann cells. Destabilization observed at some nerve terminals after extended target-deprivation, could result from the turning over of the stabilizing cues. Finally, the molecular organization of target-deprived nerve terminals was analyzed. I found that the levels of two synaptic vesicle proteins, SV-2 and synaptotagmin were reduced in target-deprived nerve terminals when compared to intact neuromuscular junctions. Analysis of cytoskeletal proteins revealed that F-actin was located at discrete bands along synaptic sites that do not colocalize with synaptic vesicle clusters. F-actin is suggested to be located at either the Schwann cell processes and/or the nerve terminal immediately above them. A possible adhesion between nerve terminals and Schwann cell processes, could contribute to the maintenance of the frog nerve terminal at the synaptic site. Finally, all target-deprived synaptic sites were found to be associated with variable levels of agrin immunoreactivity, implicating agrin as a possible maintenance molecule.

Many Oenothera species are complete translocation heterozygotes and arose by hybridization. Since complete translocation heterozygosity suppresses nuclear genetic recombination, examination of any chloroplast gene effects on hybridization is particularly suitable in these flowering plants. To verify translocation status in Oenothera villaricae, Oe. picensis ssp. picensis and lines of their interspecific hybrids, meiotic figures from parents and hybrids were obtained using confocal optical sectioning (a new application of this technique). These data confirm published results, and are consistent with a bivalent-forming common ancestor which underwent translocations to produce each of the parent species. The full translocation ring appeared in only one of the four possible hybrid nuclei. Oenothera species of hybrid origin form translocation rings at meiosis; progenitor species are structurally homozygous and form seven bivalents. Comparison of ring- and bivalent-forming species revealed hollow, abortive pollen grains in the ring-forming species (25-30% relative to 0-3% abortive pollen abortion in bivalent-forming species). F$\sb1$ hybrids of the picensis maternal lineage may abort up to 85% of pollen; nonreciprocality in the corresponding villaricae hybrid indicates nucleocytoplasmic incompatibility. Statistical analysis shows a high correlation between pollen stainability and meiotic disjunction frequency in Oe. villaricae, Oe. picensis and hybrids, suggesting that the abortive grains are the products of nondisjunctional meiosis. Significant effects (P $<$.001) on disjunction frequency and pollen stainability were noted for nucleus and cytoplasm alone and in combination, revealing an interaction or combined effect of nucleus and cytoplasm. This combined effect influences the chromosomal pairing behavior at meiosis; hence also the pollen viability. The fitness consequences of cytoplasmic hybridization intolerance (e.g., chlorosis, pollen abortion, lethality) probably affect the relative success of incipient species. Of the two cytoplasmically inherited genetic systems, the plastid genome (plastome) is the more accessible to study. Five discrete European plastid genotypes (typified by Oe. hookeri, biennis, Lamarckiana, parviflora and argillicola), as well as those of South American Oe. villaricae and picensis, have been identified by restriction fragment length polymorphisms (RFLPs) using endonucleases PvuII, SalI, KpnI and PstI. The present RFLP study employed PvuII and KpnI to analyze plastid DNAs from across subsection Munzia, the South American taxonomic group which includes Oe. villaricae and picensis, in comparison with known European plastomes. Subsect. Munzia is divided into three series: Ancestral Renneria, the younger Allochroa, and the most recently evolved, Clelandia, composed entirely of hybrids between members of Renneria and Allochroa. At least ten new plastid genotypes are noted among 28 Munzia species examined. All three series were to some degree polymorphic. In certain hybrids of known origin, comparison of the cpDNA genetic fingerprint with those of the parent species can reveal the maternal and paternal lineage, i.e. the direction of hybridization. One particular cpDNA fingerprint which predominates throughout the subsection is presumed to represent the ancestral plastome type.

While various chronic diseases and medication use have been associated with an increased risk of falling, neuromuscular deficits, due to aging or pathology, have been strongly implicated in the etiology of falls. In particular, it has been suggested that inadequate pre-innervation can result in impact forces large enough to cause femoral neck fractures, thereby leading to a fall. Less dramatically, inadequate pre-innervation may lead to an inability to compensate for potentially destabilizing forces occurring on impact. Unfortunately, little is known about the neuromuscular activation patterns present among fallers. Neuromuscular patterns were examined between 12 elderly male fallers (mean age = 74.75) and 12 elderly males (mean age = 72.75), with no history of falling. Falling status was verified through hospital records, physician's records, or spousal interview. Fallers had experienced at least one fall during the prior year, while nonfallers had no history of falling during the prior year. All subjects were free from any known neurological disorders. Neuromuscular patterns were assessed, via EMG, over three days. A step-down task was performed from heights of 10, 20, and 30 centimeters, during sighted and unsighted conditions. In addition, balance was assessed via the stork stand, tandem walk, Romberg, and get-up-and-go tests; while various strength measurements were assessed via dynamometer or EMG. The results indicate the following: (1) Fallers had significantly slower movement time than nonfallers and were more likely to display a polyphasic pre-innervation EMG pattern; (2) Fallers had significantly shorter pre-innervation times during step-downs from 10 and 20 centimeter, but this distinction failed to hold during step-downs from 30 centimeters; (3) Fallers performed more poorly on all tests of balance; (4) No statistically significant group difference could be found on any strength measure or anthropometric assessment. Consequently, in the present study population, falling status appears to be related to a slowing of the neuromuscular system. Unfortunately, the specifics or nature of that slowing remain obscure.