INVESTIGATING THE ROLE OF SEX LETHAL IN DROSOPHILA GERMLINE SEXUAL IDENTITY

Sex determination is an essential process in sexually reproducing species. Defects in sex determination lead to disorders of sex development and infertility. Therefore, studying sex determination in different species is key to improve our understanding of this important process. In Drosophila melanogaster, the way in which germ cells determine their sexual identity is different from and less understood than somatic sex determination. To improve our understanding of Drosophila germline sex determination, I investigated the molecular function of Sex lethal (Sxl), the key gene promoting female identity in the germline. Specifically, I performed an RNA expression profiling experiment (RNA-Seq) to identify targets of SXL in the germline. This RNA-Seq revealed a few hundred genes with significant changes in expression upon loss of Sxl from the germline. This data also showed that Sxl plays a largely repressive role in the germline, as a larger number of genes became upregulated in response to loss of Sxl. One such gene is PHD Finger Protein 7, which was previously shown to promote male identity in the germline. Additionally a previously uncharacterized gene, CG15930, was validated as a target of SXL in the germline. Specifically this gene is highly expressed in males and strongly repressed by SXL in females. CG15930 mutant males have reduced fertility and germline defects, suggesting this gene is necessary for proper development of the male germline. I investigated its role in germline sexual identity and found that it promotes male sexual identity in the germline. Strikingly, CG15930 is able to induce female germ cells in a male soma to adopt male fate, and go through spermatogenesis. CG15930 makes a tudor domain protein, but its function appears to differ from that of its mouse homolog, TDRD5, which is also required male-specifically in the mouse germline. Lastly, I also conducted a screen for genes with sex-specific functions in the germline. This led to the investigation of the role of discs large (dlg) in the male germline. Interestingly, loss of dlg from the germline results in severe germline depletion in males but not in females, suggesting that it has a sex-specific germline function. Further analysis revealed that dlg is required for germ cell proliferation and to maintain junctional integrity in the male germline. Altogether, the work presented in this dissertation increases our understanding of germline sexual identity and sex-specific germ cell development. It provides valuable insight into how SXL acts to promote female identity in the germline, and reveals new avenues to study the mechanistic control of germline sexual identity

Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR.

BACKGROUND:Tick-borne infections have been increasing steadily over the years, with co-infections with Borrelia burgdorferi and Babesia microti/divergens emerging as a serious health problem. B. burgdorferi is a spirochetal bacterium that causes Lyme disease while protozoan pathogens belonging to Babesia species are responsible for babesiosis. Currently used serological tests do not always detect acute Lyme disease or babesiosis, and fail to differentiate cured patients from those who get re-infected. This is a major problem for proper diagnosis particularly in regions endemic for tick-borne diseases. Microscopy based evaluation of babesiosis is confirmatory but is labor intensive and insensitive such that many asymptomatic patients remain undetected and donate blood resulting in transfusion transmitted babesiosis. RESULTS:We conducted multiplex qPCR for simultaneous diagnosis of active Lyme disease and babesiosis in 192 blood samples collected from a region endemic for both diseases. We document qPCR results obtained from testing of each sample three times to detect infection with each pathogen separately or together. Results for Lyme disease by qPCR were also compared with serological tests currently used for Lyme disease when available. Considering at least two out of three test results for consistency, 18.2% of patients tested positive for Lyme disease, 18.7% for co-infection with B. burgdorferi and B. microti and 6.3% showed only babesiosis. CONCLUSIONS:With an 80% sensitivity for detection of Lyme disease, and ability to detect co-infection with B. microti, multiplex qPCR can be employed for diagnosis of these diseases to start appropriate treatment in a timely manner

Evaluation of Nucleoside Analogs as Antimicrobials Targeting Unique Enzymes in Borrelia burgdorferi

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5′-deoxy-5′-iodoadenosine (IADO) and 5′-deoxy-5′-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease

Diagnosis of Lyme disease by qPCR and two-tier serology.

<p>Diagnosis of Lyme disease by qPCR and two-tier serology.</p

Diagnosis of <i>B</i>. <i>burgdorferi</i> infection by C6 Lyme ELISA.

<p>Histogram showing the Lyme Index distribution produced by C6 Lyme ELISA conducted on all 192 patient samples. An index ≤ 0.90 is a negative Lyme diagnosis (118 samples), an index of 0.91–1.09 is an equivocal result (7 samples), and an index ≥ 1.10 is a positive Lyme diagnosis (67 samples).</p

Diagnosis o Lyme disease by qPCR and serological tests.

<p>Diagnosis o Lyme disease by qPCR and serological tests.</p

Venn diagram summarizing the agreement in positive diagnoses of Lyme disease using different diagnostic methods.

<p><i>B</i>. <i>burgdorferi</i> infection can be detected by qPCR at a higher rate than C6 Lyme ELISA and 2-tier serological tests. There is significant overlap among all three tests.</p

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu