The Black Testament: A Portrait of Female Genealogy in the African Diaspora

The Black Testament: A Portrait of Genealogy in the African Diaspora is a hybrid work of creative nonfiction and poetry. These pieces are based on my genealogical research into my family history. The collection traces from my discoveries in the present time back through my lived experiences, the lives of my relatives, and the lives of my ancestors. The subject of the work focuses on the women in my family and the various influences they have had in my life. The collection grapples with themes such as black womanhood, diasporic existence, and complex heritage. The traveling back of genealogical research is mirrored in the collection’s reverse chronological structure

Phage-Like Streptococcus pyogenes Chromosomal Islands (SpyCI) and Mutator Phenotypes: Control by Growth State and Rescue by a SpyCI-Encoded Promoter

We recently showed that a prophage-like Streptococcus pyogenes chromosomal island (SpyCI) controls DNA mismatch repair and other repair functions in M1 genome strain SF370 by dynamic excision and reintegration into the 5′ end of mutL in response to growth, causing the cell to alternate between a wild type and mutator phenotype. Nine of the 16 completed S. pyogenes genomes contain related SpyCI integrated into the identical attachment site in mutL, and in this study we examined a number of these strains to determine whether they also had a mutator phenotype as in SF370. With the exception of M5 genome strain Manfredo, all demonstrated a mutator phenotype as compared to SpyCI-free strain NZ131. The integrase gene (int) in the SpyCIM5 contains a deletion that rendered it inactive, and this deletion predicts that Manfredo would have a pronounced mutator phenotype. Remarkably, this was found not to be the case, but rather a cryptic promoter within the int ORF was identified that ensured constitutive expression of mutL and the downstream genes encoded on the same mRNA, providing a striking example of rescue of gene function following decay of a mobile genetic element. The frequent occurrence of SpyCI in the group A streptococci may facilitate bacterial survival by conferring an inducible mutator phenotype that promotes adaptation in the face of environmental challenges or host immunity

The Bacterial Type III Secretion System as a Target for Developing New Antibiotics

Antibiotic resistance in pathogens requires new targets for developing novel antibacterials. The bacterial type III secretion system (T3SS) is an attractive target for developing antibacterials as it is essential in the pathogenesis of many Gram-negative bacteria. The T3SS consists of structural proteins, effectors and chaperones. Over 20 different structural proteins assemble into a complex nanoinjector that punctures a hole on the eukaryotic cell membrane to allow the delivery of effectors directly into the host cell cytoplasm. Defects in the assembly and function of the T3SS render bacteria non-infective. Two major classes of small molecules, salicylidene acylhydrazides and thiazolidinones, have been shown to inhibit multiple genera of bacteria through the T3SS. Many additional chemically and structurally diverse classes of small molecule inhibitors of the T3SS have been identified as well. While specific targets within the T3SS of a few inhibitors have been suggested, the vast majority of specific protein targets within the T3SS remain to be identified or characterized. Other T3SS inhibitors include polymers, proteins and polypeptides mimics. In addition, T3SS activity is regulated by its interaction with biologically relevant molecules, such as bile salts and sterols, which could serve as scaffolds for drug design

Lexicographic ordering: intuitive multicriteria optimization for IMRT

Optimization problems in IMRT inverse planning are inherently multicriterial since they involve multiple planning goals for targets and their neighbouring critical tissue structures. Clinical decisions are generally required, based on tradeoffs among these goals. Since the tradeoffs cannot be quantitatively determined prior to optimization, the decision-making process is usually indirect and iterative, requiring many repetitive optimizations. This situation becomes even more challenging for cases with a large number of planning goals. To address this challenge, a multicriteria optimization strategy called lexicographic ordering (LO) has been implemented and evaluated for IMRT planning. The LO approach is a hierarchical method in which the planning goals are categorized into different priority levels and a sequence of sub-optimization problems is solved in order of priority. This prioritization concept is demonstrated using two clinical cases (a simple prostate case and a relatively complex head and neck case). In addition, a unique feature of LO in a decision support role is discussed. We demonstrate that a comprehensive list of planning goals (e.g., ∼23 for the head and neck case) can be optimized using only a few priority levels. Tradeoffs between different levels have been successfully prohibited using the LO method, making the large size problem representations simpler and more manageable. Optimization time needed for each level was practical, ranging from ∼26 s to ∼217 s. Using prioritization, the LO approach mimics the mental process often used by physicians as they make decisions handling the various conflicting planning goals. This method produces encouraging results for difficult IMRT planning cases in a highly intuitive manner.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58100/2/pmb7_7_006.pd

ELUCIDATION OF THE NEEDLE-TIP AND TIP-TRANSLOCON INTERACTIONS OF THE SALMONELLA SPI-1 TYPE III SECRETION SYSTEM AND IDENTIFICATION OF SMALL MOLECULE BINDERS OF THE TIP AND TRANSLOCON PROTEINS

The type III secretion system (T3SS) is required by many pathogenic Gram-negative bacteria for the initiation and maintenance of infections within eukaryotic host cells. T3SS harboring bacteria include the causative agents of food poisoning/typhoid fever (Salmonella Typhimurium/Typhi), dysentery (Shigella flexneri/dysenteriae), nosocomial pneumonia (Pseudomonas aeruginosa), bubonic plague (Yersinia pestis), melioidosis (Burkholderia pseudomallei), and trachoma (Chlamydia trachomatis). Together, these bacteria are estimated to result in millions of deaths worldwide each year. Therefore, it is of great interest to elucidate the mechanisms of T3SS-mediated virulence utilized by pathogenic Gram-negative bacteria. Salmonella is the focus of this dissertation because it is an excellent model organism for T3SS research due to the ease of genetic manipulation and the availability of biological assays.The T3SS is utilized to inject bacterial virulence factors (also known as effectors) into the host cell cytoplasm, where they manipulate host cell signaling pathways to promote bacterial engulfment, maintenance of infection, and evasion of the host immune system. T3SS effectors are translocated across both bacterial and host cell membranes by the structural component of the T3SS, the needle apparatus. The needle apparatus contains a bacterial membrane embedded base structure, an extracellular needle with a 25A wide channel, a tip complex that regulates secretion and serves as an environmental sensor, and translocon proteins that assemble a pore in the host cell membrane. How the needle, tip and translocon proteins interact with each other to assemble a functional T3SS needle apparatus and coordinate the secretion of T3SS effectors is poorly understood. Because the needle, tip and translocon proteins are essential for the pathogenesis of T3SS harboring bacteria, are exposed to the extracellular environment during infection, and are conserved in structure and function, they are attractive targets for the development of novel virulence based anti-bacterial therapeutics. Hence, the importance of elucidating the structure, function and molecular interactions of the T3SS needle, tip and translocon proteins.This dissertation is focused on two major themes. The first theme is the elucidation of essential protein-protein interactions of the Salmonella T3SS needle apparatus through a combination of solution nuclear magnetic resonance (NMR) and fluorescence spectroscopy. To this end, I used amide (15N) and isoleucine, leucine and valine methyl (ILV 13C-methyl) probes in NMR titrations to map the interaction of T3SS proteins. I additionally labeled T3SS proteins with fluorescent probes to perform fluorescence polarization (FP) and Förster resonance energy transfer (FRET) protein- protein binding assays to complement the NMR studies. Using these methods, the interaction between the Salmonella SP-1 T3SS needle protein PrgI and the tip protein SipD, as well as between SipD and the major translocon protein SipB, are described in detail and validated using bacterial invasion assays. The results of NMR and FP/FRET experiments allowed for the proposal of a model for the needle/tip/translocon protein- protein interaction interface where the proximal end of SipD (the bottom of the coiled- coil) is used for interaction with PrgI, while the distal end of SipD (the top of the coiled- coil and the mixed α/β domain) is the surface used for interaction with SipB. The second theme is focused on the T3SS needle apparatus as an attractive target for the development of inhibitors. A review of the current T3SS inhibitor literature is described. In addition, I identified small molecules binders of the tip and translocon proteins from a surfaceivplasmon resonance (SPR) screen and subsequently validated and mapped the protein- small molecule interactions using titration and saturation transfer different (STD) NMR spectroscopy