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Articles 1 - 30 of 90
Full-Text Articles in Life Sciences
Protein Trafficking In The Endoplasmic Reticulum Mediated By A Tpr-Containing Adapter Protein, Nathan P. Canniff
Protein Trafficking In The Endoplasmic Reticulum Mediated By A Tpr-Containing Adapter Protein, Nathan P. Canniff
Doctoral Dissertations
The endoplasmic reticulum (ER) is a large, multifunctional organelle that acts as the entrance into the secretory pathway, which accounts for the folding and maturation of approximately one third of the human proteome. It is the largest organelle in most cell types and is comprised of a single lumen and a contiguous membrane. The ER is responsible for a multitude of roles including protein translocation, folding, maturation, quality control, and glycosylation to name a few. These processes are buoyed by a large collection of chaperones and cochaperones, the largest subfamily of which is characterized by the presence of tetratricopeptide repeat …
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Combining Simulation And The Mspa Nanopore To Study P53 Dynamics And Interactions, Samantha A. Schultz
Masters Theses
p53 is a transcription factor and an important tumor suppressor protein that becomes activated due to DNA damage. Because of its role as a tumor suppressor, mutations in the gene that encodes it are found in over 50% of human cancers. The N-terminal transactivation domain (NTAD) of p53 is intrinsically disordered and modulates the function and interactions of p53 in the cell. Its disordered structure allows it to be controlled closely by post-translation modifications that regulate p53’s ability to bind DNA and interact with regulatory binding partners. p53 is an attractive target for developing cancer therapeutics, but its intrinsically disordered …
A Shift In Rna Fate: Investigating The Role Of C19orf66 During Kshv Lytic Replication, William Rodriguez Jr.
A Shift In Rna Fate: Investigating The Role Of C19orf66 During Kshv Lytic Replication, William Rodriguez Jr.
Doctoral Dissertations
During viral infection, virus and host clash for control of the cell in a conflict that ultimately drives the evolution of both sides and has lasting consequences in the form of pathogenesis. At the heart of this struggle is a contest for control of cellular gene expression, a struggle epitomized by an evolutionary tug-of-war for supremacy over RNA fate. During lytic replication, Kaposi’s sarcoma-associated herpesvirus (KSHV) triggers a massive RNA decay event known as viral “Host Shutoff” which decimates greater than 70% of the host transcriptome, simultaneously suppressing the cellular anti-viral response and freeing host resources for viral replication. However, …
Modulation Of Plant Immunity During The Establishment Of The Nitrogen-Fixing Symbiosis, Miriam Hernandez-Romero
Modulation Of Plant Immunity During The Establishment Of The Nitrogen-Fixing Symbiosis, Miriam Hernandez-Romero
Doctoral Dissertations
Nitrogen is essential for plant tissue growth but is often a limited resource in soils. Many legumes overcome this limitation by entering a symbiotic association with soil microbes, called rhizobia, which provide nitrogen to the plant while rhizobia receive fixed carbon. To successfully form a symbiosis, the host and symbiont exchange a series of molecular signals. One major obstacle during this interaction is the host's innate immune system, which becomes active upon rhizobial detection. It is therefore the main focus of this thesis to identify the mechanisms that modulate host immunity. In the subsequent chapters, we focus on a rhizobial …
Changes In Gene Expression From Long-Term Warming Revealed Using Metatranscriptome Mapping To Fac-Sorted Bacteria, Christopher A. Colvin
Changes In Gene Expression From Long-Term Warming Revealed Using Metatranscriptome Mapping To Fac-Sorted Bacteria, Christopher A. Colvin
Masters Theses
Soil microbiomes play pivotal roles to the health of the environment by maintaining metabolic cycles. One question is how will climate change affect soil bacteria over time and what could the repercussions be. To answer these questions, the Harvard Forest Long-Term Warming Experiment was established to mimic predicted climate change by warming plots of land 5℃ above ambient conditions. In 2017, 14 soil core samples were collected from Barre Woods warming experiment to mark 15 years since the establishment of the soil warming in that location. These samples underwent traditional metatranscriptomics to generate an mRNA library as well as a …
Characterization Of The Poly (Adp-Ribose) Polymerase Family In The Fusarium Oxysporum Species Complex, Daniel Norment
Characterization Of The Poly (Adp-Ribose) Polymerase Family In The Fusarium Oxysporum Species Complex, Daniel Norment
Masters Theses
Fusarium oxysporum is a filamentous fungus that is known to invade over a hundred different hosts and poses a major threat to the economy and food supply world-wide. Poly (Adenosine diphosphate-Ribose) Polymerase (PARP) is a family of regulatory proteins that affect change in the cell through transfer of ADP-Ribose moieties onto target molecules. The most well-studied PARP protein is the human PARP1, a PARylating nuclear protein that serves as our model PARP protein. F. oxysporum was found to contain a large expansion of PARP catalytic-domain-containing proteins compared to other filamentous fungi. We utilized in silico multiple sequence alignments and domain …
Chemosensory Receptors In Berghia Stephanieae: Bioinformatics And Localization, Kelsi L. Watkins
Chemosensory Receptors In Berghia Stephanieae: Bioinformatics And Localization, Kelsi L. Watkins
Masters Theses
Chemosensation is achieved through the binding of chemical signals to chemoreceptor proteins embedded in the membranes of sensory neurons. The molecular identity of these receptors, as well as the downstream processing of chemosensory signals, has been well studied in arthropods and vertebrates. However, very little is known about molluscan chemosensation. The identity of chemoreceptor proteins in the nudibranch mollusc Berghia stephanieae are unknown. Data from other protostome and molluscan studies suggest Berghia may use ionotropic receptors for some forms of chemoreception. This study used a bioinformatics approach to identify potential chemosensory ionotropic receptors in the transcriptome of Berghia. A …
The Sos Response In Escherichia Coli K12: An Exploration Of Mutations In Lexa And Reca Using Fluorescence Microscopy, Steven Van Alstine
The Sos Response In Escherichia Coli K12: An Exploration Of Mutations In Lexa And Reca Using Fluorescence Microscopy, Steven Van Alstine
Doctoral Dissertations
Faithful replication of the genome is paramount for maintaining the fitness of an organism. Therefore, life has evolved inducible mechanisms to be able to repair damaged DNA and maintain evolutionary fitness. The SOS response is a highly conserved DNA damage inducible response that is tightly regulated. Multiple factors contribute to the ability of the cell to perform proper DNA repair and induction of the SOS response including the amount of RecA, mutations in RecA that affect competition for DNA, and other proteins that interact with the RecA filament. The complex relationship between RecA and LexA is the subject of this …
Chemical Biology Approaches For Tracking And Manipulation Of Macrophage Phenotypes, Javier A. Mas Rosario
Chemical Biology Approaches For Tracking And Manipulation Of Macrophage Phenotypes, Javier A. Mas Rosario
Doctoral Dissertations
Macrophages are white blood cells of the innate immune system that have the ability to change phenotypically depending on the stimuli present in their surroundings through a process commonly referred to as polarization. Macrophage phenotypes broadly range from pro-inflammatory, anti-tumor (M1) to immune-suppressing (M2). Of particular interest to this work, breast cancer progression and metastasis rely on the presence of M2-like tumor-associated macrophages (TAMs). While many studies have shown the involvement of macrophages in tumor progression and metastasis, there remains a need to further explore these interactions and the polarization process, including tracking of macrophage subtypes. Toward this end, I …
Characterizing The Multifaceted Roles Of The Proteasomal Deubiquitinase Uch37 In Proteostasis, Heather A. Bisbee
Characterizing The Multifaceted Roles Of The Proteasomal Deubiquitinase Uch37 In Proteostasis, Heather A. Bisbee
Doctoral Dissertations
Cellular protein pools are maintained through the biological processes of synthesis, degradation and quality control. As the dysregulation of these processes has been implicated in diseases such as neurodegeneration and cancer, understanding their functions is critical for drug development. Modification of proteins with ubiquitin may direct them to the proteasome, a large cellular protease complex, for degradation. Yet, the proteasome contains three deubiquitinating enzymes (DUBs) which remove ubiquitin from proteins, potentially altering their fate. As each DUB recognizes specific ubiquitin linkages and architectures, their activity may regulate how the proteasome handles substrates in dynamic cellular contexts. In this work, we …
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Controlling Myosin’S Function Via Interactions Between The Substrate And The Active Site, Mike K. Woodward
Doctoral Dissertations
Molecular motors, such as myosin, have evolved to transduce chemical energy from ATP into mechanical work to drive essential cellular processes, from muscle contraction to vesicular transport. Dysfunction in these motors is a root cause of many pathologies necessitating the application of intrinsic control over molecular motor function. We hypothesized that altering the myosin’s energy substrate via minor positional changes to the triphosphate portion of the molecule will allow us to control the protein and affect its in vitro function. We utilized positional isomers of a synthetic non-nucleoside triphosphate, azobenzene triphosphate, and assessed whether myosin’s force- and motion-generating capacity could …
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Investigating Structures And Functions Of Apoptotic Caspases, Ishankumar V. Soni
Doctoral Dissertations
Caspases are cysteine aspartate proteases involved in various cellular pathways including apoptosis, inflammation, and neurodegeneration. Caspase-9 is classified as an initiator apoptotic caspase that is activated upon intrinsic stress. Caspase-9 is composed of two domains: an N- terminal CARD domain and a catalytic core domain. We have employed hydrogen deuterium exchange mass spectrometry (H/DX-MS) to determine the 1) dynamics of the full-length caspase- 9, 2) dynamic impacts on caspase-9 upon substrate-induced dimerization, and 3) regions involved in the CARD: catalytic core domains interactions. Upon intrinsic stress, caspase-9 activates executioners, procaspase-3 and -7 but not procaspase-6. We have employed site-directed mutagenesis …
Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don
Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don
Doctoral Dissertations
Circadian rhythms are 24–hour activity cycles that exist for all organisms, from yeast and bacteria to mammals. Epidemiological data has shown that disruptions to circadian rhythms are associated with various diseases, including cancers. Circadian rhythms are regulated at the cellular level by a "molecular clock,” whose oscillations in protein transcription and translation control a range of downstream pathways. However, the connections between altered rhythms and diseases at the molecular level is unclear. Therefore, in this project, I tracked circadian rhythms in a high-resolution manner and thoroughly analyzed them to more completely reflect circadian behaviors and clock functioning at the molecular …
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Masters Theses
Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a …
Characterizing Mechanical Regulation Of Bone Metastatic Breast Cancer Cells, Boyuan Liu
Characterizing Mechanical Regulation Of Bone Metastatic Breast Cancer Cells, Boyuan Liu
Doctoral Dissertations
Breast cancer most frequently metastasizes to the skeleton. Bone metastatic cancer is incurable and induces wide-spread bone osteolysis, resulting in significant patient morbidity and mortality. Mechanical stimuli in the skeleton are an important microenvironmental parameter that modulates tumor formation, osteolysis, and tumor cell-bone cell signaling, but which mechanical signals are the most beneficial and the corresponding molecular mechanisms are unknown. This work focused on bone matrix deformation and interstitial fluid flow based on their well-known roles in bone remodeling and in primary breast cancer. The goal of our research was to establish a platform that could define the relationship between …
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Quantitative Imaging Of Tensile Forces At Cell-Cell Junction With Dna-Based Probes, Puspam Keshri
Doctoral Dissertations
Mechanical forces are an integral part in biology, they regulate several cellular properties, such as morphology, proliferation, migration. These forces are also involved in receptor signaling and the differentiation of different cell types. Different proteins and biomolecules such as cadherin, integrin, notch proteins are essential elements of these processes. Measuring these intercellular forces are challenging considering the minimal intensity (piconewton-level) of these molecular forces. In our lab, we have developed a membrane DNA tension probe (MDTP) that uses a DNA hairpin module to sense tensile forces and has a lipid anchor to modify onto live-cell membranes. The programmability of DNA …
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Understanding How Camkii Holoenzyme Dynamics Facilities Activation-Triggered Subunit Exchange, Ana P. Torres-Ocampo
Doctoral Dissertations
Long-term memory and learning are still poorly understood from a molecular and cellular standpoint. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an oligomeric kinase that is involved in this remarkable process. However, the molecular details of its specific roles in these processes remains elusive. CaMKII activation-triggered subunit exchange presents a novel possible mechanism involved in long-term memory and learning by exchanging active subunits with other CaMKIIs. CaMKII subunit exchange also shows that exchanged CaMKIIs spread their phosphorylation state to newly synthesized CaMKIIs. This provides a long-lasting signal that might possibly be involved in long-term memory by escaping a cell’s …
Structure-Function Studies Of The Trypanosome Mitochondrial Replication Protein Polib, Raveen Armstrong
Structure-Function Studies Of The Trypanosome Mitochondrial Replication Protein Polib, Raveen Armstrong
Masters Theses
Trypanosoma brucei and related protists are distinguished from all other eukaryotes by an unusual mitochondrial genome known as kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Replication of this single nucleoid involves a release, replicate, and reattach mechanism for the thousands of catenated minicircles and requires at least three DNA polymerase (POLIB, POLIC and POLID) with similarity to E. coli DNA polymerase I. Like other proofreading replicative DNA polymerases, POLIB has both an annotated polymerase domain and an exonuclease domain. Predictive modelling of POLIB indicates that it has the canonical right hand …
Utilizing Fluorescence Microscopy To Characterize The Subcellular Distribution Of The Novel Protein Acheron, Varun Sheel
Utilizing Fluorescence Microscopy To Characterize The Subcellular Distribution Of The Novel Protein Acheron, Varun Sheel
Masters Theses
All cells carry the genetic machinery required to commit cell suicide; a process known as programmed cell death (PCD). While the ability to initiate PCD serves a number of useful purposes during development and homeostasis, misregulation of PCD is the underlying basis of most human diseases, including cancer, autoimmunity disorders and neurodegeneration. Using the tobacco hawkmoth Manduca sexta as a model organism, the Schwartz lab at UMass has demonstrated that PCD requires de novo gene expression and has cloned many death-associated genes. One of these genes encodes a novel protein that was named Acheron after one of the rivers of …
Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini
Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini
Masters Theses
Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, …
Exploring Knockdown Phenotypes And Interactions Between Atad3 Proteins In Arabidopsis Thaliana, Eli S. Gordon
Exploring Knockdown Phenotypes And Interactions Between Atad3 Proteins In Arabidopsis Thaliana, Eli S. Gordon
Masters Theses
Mitochondria are required for a diverse array of cellular functions and processes. ATAD3 (ATPase family AAA domain containing protein 3) proteins are newly discovered mitochondrial membrane proteins in Arabidopsis thaliana. Homologous to ATAD3A in metazoans, Co-Immunoprecipitation/Mass spectrometry and genomic analysis identified a four ATAD3A homologues in A. thaliana. The four A. thaliana proteins are referred to as ATAD3A1 (At3g03060), ATAD3A2 (At5g16930), ATAD3B1 (At2g18330), and ATAD3B2 (At4g36580). Studies in metazoans indicate that ATAD3A localizes to Mitochondria-ER contact sites and is involved in a variety of processes required for proper mitochondrial function, but ATAD3A proteins are poorly defined in plants. …
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Novel Approaches Towards Improved Purity In High Yield Transcription Reactions, Elvan Cavac
Doctoral Dissertations
High yields of RNA (e.g., mRNA, gRNA, lncRNA) are routinely prepared following a two-step approach: high yield in vitro transcription using T7 RNA polymerase, followed by extensive purification using gel or chromatic methods. In high yield transcription reactions, as RNA accumulates in solution, T7 RNA polymerase rebinds and extends the encoded RNA (using the RNA as a template), resulting in a product pool contaminated with longer than desired, (partially) double stranded impurities. Current purification methods often fail to fully eliminate these impurities which, if present in therapeutics, can stimulate the innate immune response with potentially fatal consequences. This study establishes …
Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu
Pharmacological Chaperoning Of Human Lysosomal Neuraminidase 1, Di Chu
Doctoral Dissertations
Human lysosomal neuraminidase 1 (hNEU1) is an exo-a-sialidase which cleaves a(2-3) and a(2-6) linked sialic acids on glycoproteins in the lysosome. Deficiency of hNEU1 in the lysosome results in sialidosis, a lysosomal storage disease. Currently there is no effective treatment for sialidosis, which leads to a rising interest in discovering potential therapies. Here we presented a small molecule, α-D-N-acetylneuraminic acid (NANA), increases the protein amount and activity of both wild-type hNEU1 and three different hNEU1 mutations found in sialidosis patients in our mammalian cell system, suggesting that NANA works as a potential pharmacological chaperone for hNEU1 and provides …
Investigation Of Shot1-Binding Atpases In Arabidopsis Thaliana, Sam Zelman
Investigation Of Shot1-Binding Atpases In Arabidopsis Thaliana, Sam Zelman
Masters Theses
Mitochondria play critical roles not only in primary metabolism as a central organelle for ATP generation, but also in responding to abiotic stresses. We identified a mutation in the MTERF18 (Mitochondrial Transcription Termination factor)/SHOT1 (Suppressor of hot1-4 1) gene in Arabidopsis thaliana that enables plants to better tolerate heat and oxidative stresses, presumably due to reduced oxidative damage, but the exact molecular mechanism of the heat tolerance is unknown. In order to reveal the stress tolerance mechanisms of mterf18/shot1 mutations, it is critical to understand the molecular defects of the mutant and to identify …
Exploration Of The Association Between Muscle Volume And Bone Geometry Reveals Surprising Relationship At The Genetic Level, Prakrit Subba
Exploration Of The Association Between Muscle Volume And Bone Geometry Reveals Surprising Relationship At The Genetic Level, Prakrit Subba
Masters Theses
The evolution of jaws in cichlid fishes of the East African Great Lakes is a textbook example of adaptive radiation in vertebrates. Karl Liem postulated that this adaptive radiation has been possible due to the functional decoupling of two cichlid functional units – the pharyngeal jaw (PJ) and the oral jaw (OJ). This functional decoupling of the jaws has enabled the OJ to be relieved of its dual role of prey capturing and processing and has allowed the PJ to take on the role of prey processing. As a result, African cichlids have adapted the morphology of their functional units …
Assessing Stress Tolerance Of Organelle Small Heat Shock Protein Mutants In Arabidopsis Thaliana, Parth Patel
Assessing Stress Tolerance Of Organelle Small Heat Shock Protein Mutants In Arabidopsis Thaliana, Parth Patel
Masters Theses
Molecular chaperones are proteins found in virtually every organism and are essential to cell survival. When plants are heat stressed, they upregulate and downregulate multiple genes, many of which are associated with the heat shock response. Small heat shock proteins (sHSPs) are one class of molecular chaperones that are upregulated during heat shock. They are proposed to act as the first line of defense by binding to heat sensitive proteins and preventing their irreversible aggregation. However, many details of sHSP function remain to be discovered and exactly what proteins they protect is unresolved. In addition to cytosolic sHSPs found in …
The Shape Of U: Mapping Out Protective Elements In Mrna Escapees, Jacob Miles
The Shape Of U: Mapping Out Protective Elements In Mrna Escapees, Jacob Miles
Masters Theses
A crucial step of the viral life cycle of Kaposi’s Sarcoma Herpesvirus (KSHV) lytic infection is the triggering of a massive RNA decay event termed “Host Shutoff”. Host Shutoff is driven by the viral endonuclease SOX which leads to the destruction of over 70% of the total transcriptome. This process cripples cellular gene expression and allows for viral reprograming of the cell for the purpose of viral replication. Co-evolution has led to the host developing a multitude of antiviral defenses aimed at preserving certain cellular RNAs linked to antiviral responses. One such defense are RNA secondary structures located within the …
Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois
Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois
Doctoral Dissertations
Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Doctoral Dissertations
Many legumes form a symbiosis with nitrogen-fixing bacteria found in the soil. This relationship is beneficial to both the plant and the bacteria; the plant receives nitrogen that is otherwise limited, and the bacteria receive fixed carbon. Upon sensing the bacteria, the plant forms a new organ (the nodule) where the bacteria are housed within the cells. Many genes are required for the proper formation and function of nodules; this dissertation is broadly focused on how genes required for nitrogen-fixing symbiosis are co-opted from other cellular processes and how they are specialized for symbiosis. Protein trafficking from the plant to …
Structure Function Analysis Of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles In Kinetoplast Maintenance, Jonathan Miller
Structure Function Analysis Of Mitochondrial Dna Polymerase Ic Reveals Multiple Roles In Kinetoplast Maintenance, Jonathan Miller
Doctoral Dissertations
Kinetoplastid organisms including medically relevant species like Trypanosoma brucei, Trypanosoma cruzi, and Leishmania are distinguished by their single flagellum and unique mitochondrial DNA called kDNA, among other features. While there is heterogeneity in copy number and sequence classes among different species, kDNA is most often found as a network of thousands of DNA molecules catenated together. This unique biological property of disease-causing organisms has been a subject of study as a potential drug target since it is essential for parasite survival. Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and related diseases in other mammals and …