Over a billion individuals worldwide suffer from neglected diseases. This equates to approximately one-sixth of the human population. These infections are often endemic in remote tropical regions of impoverished populations where vectors can flourish and infected individuals cannot be effectively treated due to a lack of hospitals, medical equipment, drugs, and trained personnel. The few drugs that have been approved for the treatments of such illnesses are not widely used because they are riddled with inadequate implications of cost, safety, drug availability, administration, and resistance. Hence, there exists an eminent need for the design and development of improved new therapeutics. Influential world-renowned scientists in the Consortium for Parasitic Drug Development (CPDD) have preformed extensive biological testing for compounds active against parasites that cause neglected diseases. These data were acquired through several collaborations and found applicable to computational studies that examine quantitative structure-activity relationships through the development of predictive models and explore structural relationships through docking. Both of these in silico tools can contribute to an understanding of compound structural importance for specific targets. The compilation of manuscripts presented in this dissertation focus on three neglected diseases: trypanosomiasis, Chagas disease, and leishmaniasis. These diseases are caused by kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., respectively. Statistically significant predictive devices were developed for the inhibition of the: (1) T. brucei P2 nucleoside transporter, (2) T. cruzi parasite at two temperatures, and (3) two species of Leishmania. From these studies compound structural importance was assessed for the targeting of each parasitic system. Since these three parasites are all from the Order Kinetoplastida and the kinetoplast DNA has been determined a viable target, compound interactions with DNA were explored to gain insight into binding modes of known and novel compounds.

Leishmaniasis is caused by infection with protozoan parasites within the genus Leishmania and, in the New World, is transmitted by the bites of female sand flies within the genus Lutzomyia. The occurrence of leishmaniasis in rodent species, the geographic distribution of sand fly species in Nicaragua, and environmental factors associated with the distribution of human cases of typical cutaneous leishmaniasis were investigated. Three hundred ninety five rodents representing 17 species were collected from 13 localities from August 2001–March 2006 and screened for Leishmania infections. One Heteromys desmarestianus and one Peromyscus mexicanus were found to be positive for leishmanial infections by PCR. This is the first report of Leishmania infections in rodents in Nicaragua. Five hundred fifty six sand flies representing 12 species were collected from 8 localities, including Lutzomyia hartmanni, a new record for this species in Nicaragua. The predominant sand fly species captured in western Nicaragua were Lutzomyia longipalpis and Lutzomyia evansi. The predominant species captured in central and eastern Nicaragua was Lutzomyia cruciata. The geographic distribution of sand flies in this study provides additional support to previouslypublished reports of suspected vectors of Leishmania species that cause typical and atypical forms of cutaneous leishmaniasis in Nicaragua. Distribution data of human cases of typical cutaneous leishmaniasis obtained from the Nicaraguan Ministry of Health, along with GIS and remotely sensed data of elevation, precipitation, temperature, soil types and land use/cover classes, were used to develop predictive logistic regression models for the presence or absence of human cases within 151 municipalities. Mean annual precipitation and land use/cover were determined to be the best environmental variable predictors for the occurrence of typical cutaneous leishmaniasis.

Altered cytoskeletal regulation impacts numerous physiological phenomena: cell motility, apoptosis, oncogenic transformation and parasitic infection. The protein tyrosine phosphatase (PTP)-PEST contains multiple motifs mediating its recruitment to signalling components, and is required for actin filament organization. However, little is known regarding either the importance of PTP-PEST subcellular localization, or the role of PTP-PEST in either parasitic infection or apoptosis. My doctoral research was therefore focussed on elucidating the effect of subcellular distribution on PTP-PEST activity, specifically with respect to regulation of p130Cas (a PTP-PEST substrate), as well as on the involvement of PTP-PEST in both host-pathogen relations and apoptosis. First, PTP-PEST was found both within the cytosol and at the plasma membrane. Using PTP-PEST -/- rescued cell lines, I observed that tyrosine phosphorylation-dependent p130Cas interactions were controlled primarily by cytosolic PTP-PEST. Secondly, infection of fibroblasts with Leishmania major was observed to induce dramatic actin rearrangements, and to alter the phosphorylation state of numerous proteins. Importantly, both PTP-PEST and p130Cas were processed by the parasitic protease GP63 during infection. GP63 was also required for the cleavage of additional host proteins: cortactin, TC-PTP and caspase-3. Of note, Leishmania parasites mediated p38 inactivation, correlating with the proteolysis of its upstream activator TAB1, in a GP63dependent manner. These results indicate that GP63 plays a key role in a number of biochemical events, potentially contributing to Leishmania infectivity. Finally, PTP-PEST was found to relocalize to the edges of retracting membrane ruffles of apoptotic cells. Surprisingly, PTP-PEST was specifically cleaved by caspase-3 at the 549DSPD motif during apoptosis; leading to modification of catalytic activity and scaffolding properties, and sensitizing cells to Fas-mediated detachment. As this data demonstrated a potential role for caspase cleavage in PTP regulation, I also investigated the presence of conserved putative caspase-c1eavage sites in other family members. In summary, the data presented herein links PTP-PEST with various biological processes: oncogenic signalling, host-pathogen interactions, and apoptosis. In addition to demonstrating the involvement of PTP-PEST in diverse signalling pathways, these studies underscore the importance of subcellular localization and proteolysis in the regulation of this PTP.

In Leishmania glycosomes compartmentalize a variety of essential biochemical and metabolic pathways required for parasite viability. Biogenesis and maintenance of glycosomes involves a family of proteins called peroxins, however the molecular mechanisms governing the early events of this pathway have not been fully established. / A structural-functional analysis of the receptor LdPEX5 revealed the formation of a tetrameric structure stabilized by coiled-coil motifs. A biophysical approach showed that the tetrameric structure of LdPEX5 dissociates to dimers upon binding to the PTS1 ligand. However, the tetrameric LdPEX5 is more thermodynamically favorable to bind. Lastly, LdPEX14 modulates the LdPEX5-PTS1 interaction as the presence of LdPEX14 decreases the Kd of LdPEX5-PTS1 by ∼10 folds. / PTS1-loaded LdPEX5 docks onto the glycosomal membrane via the membrane-associated LdPEX14, an interaction that was characterized by molecular mapping and biophysical approaches. In higher eukaryotes this PEX5-PEX14 interaction involves conserved WXXXF/Y pentapeptide motifs found on PEX5 and a signature sequence found on PEX14. These three repeats in LdPEX5 do not appear to be crucial for interaction with LdPEX14 thus suggesting a unique molecular mechanism mediating the docking event. On the other hand, the conserved signature motif is crucial for interaction with LdPEX5. / The topology and nature of the interaction of LdPEX14 with the glycosomal membrane is not clear. In vivo expression of FLAG-LdPEX14-HA together with proteinase digestion confirmed that both N- and C-termini are cytosolic facing. Mapping analysis revealed that the first 63 N-terminal residues of LdPEX14 are critical for anchoring LdPEX14 to the glycosomal membrane. Interestingly, expression of the first 74 amino acids of LdPEX14 is toxic to the parasites. / Finally, the structure of LdPEX14 on the glycosomal membrane was addressed by molecular mapping and biophysical techniques. Partial trypsin digestion of recombinant LdPEX14 and molecular mapping suggested that the first 250 residues of LdPEX14 were involved in the formation of the complex. Biophysical approaches and cross-linking assays suggest that this complex may consist of ∼12-100 LdPEX14 subunits. Interestingly, the structure of LdPEX14 appears to be modulated by LdPEX5. / Considering all the results, these findings have established important molecular information concerning early events in the targeting and import of PTS1 proteins into the glycosome.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 175-196).

Orientador: Jairo Aparecido Ayres / Banca: Maria de Lourdes da Silva Marques Ferreira / Banca: Roseli Moralez de Figueiredo / Resumo: A constante busca do progresso em virtude da globalização tem contribuído para causar alterações no equilíbrio biológico, social e cultural, repercutindo diretamente no processo saúde-doença da população. Neste contexto, destaca-se as doenças emergentes, dentre estas tem-se a Leishmaniose Visceral como problema de saúde publica com alta letalidade quando não diagnosticada e tratada precocemente. Na Divisão Regional de Saúde VI Bauru (DRS VI Bauru) entre os anos de 1999 e 2009 ocorreram 417 casos da doença, sendo que, 41 destes evoluíram para óbitos. Por estas informações sobre a LV, despertou interesse em estudar os aspectos sociodemografico, estabelecer os diagnósticos de enfermagem e sistematizar a assistência. Para tanto, no período estudado foram realizadas 22 entrevista semi-estruturadas com portadores, internados em hospital de referencia. Estes, eram adultos procedentes de Bauru - SP e região, de ambos os sexos, sendo 15 do sexo masculino (68,19%) e sete do sexo feminino (31,81%), na faixa etária de 20 a 66 anos, com prevalência do 1º grau incompleto (16 casos - 72,74%) e renda familiar entre um a dois salários mínimos (10 casos - 45,45%), revelavam comprometimento sistêmico compatível com LV clássica. Foi aplicado o Processo de Enfermagem para identificar os principais Diagnostico de Enfermagem segundo NANDA e propor plano assistencial. Por estes dados, possibilitou conhecer os aspectos epidemiológicos, fisiopatológicos e terapêuticos da LV, assim como Diagnósticos de Enfermagem, que possibilitaram estabelecer ações independentes, dependentes e interdependentes, proporcionando o bem estar, com olhar diferenciado da assistência individualizada e integralizada aos portadores desta doença. Acredita-se também que com este estudo foi possível destacar alem do papel assistencial e a ação educacional do enfermeiro, que ocorreu durante... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The constant search for progress because of globalization has contributed to cause changes in the biological, social and cultural balance, with significant repercussions on the health-disease population. In this context, the emerging diseases stands out, among them, the Visceral Leishmaniasis, as public health problem with high mortality if not diagnosed and treated early. In Division VI Regional Health Bauru (Bauru DRH VI) between the years 1999 and 2009 there were 417 cases of the disease, of which 41 of these ones, progressed to deaths. For information about VL, interests were aroused in studying the sociodemographic characteristics, establish nursing diagnoses and systematize assistance. To this end, during the study period there were 22 semi-structured interviews with patients hospitalized in a reference hospital. These patients were adults coming from Bauru - SP region, of both sexes, being 15 males (68.19%) and seven females (31.81%), aged between 20 to 66 years old, with prevalence of an incomplete degree (16 cases - 72.74%) and family income between one and two minimum wages (10 cases - 45.45%) showed systemic involvement consistent with classic VL. The nursing process was applied and identified the main Nursing Diagnosis according to NANDA, and care planning was proposed. For these data, the epidemiology, pathophysiology and treatment of VL were better known, as well as nursing diagnoses, which enabled the establishment of independent, dependent and interdependent actions that, giving the well-being, with a fully different individual care view to the disease patients. It is also believed that this study could highlight beyond the action role and the nurses care and education, which occurred during the hospitalization and interactions period, even not being an objective of study, showed its importance for the ill clarification, since it evidenced some unfamiliarity about... (Complete abstract click electronic access below) / Mestre

Publicación a texto completo no autorizada por el autor / Evalúa la capacidad mitogénica del extracto de lectinas de semillas de tarwi del ecotipo “Patón Grande” (UNALM-Otuzco, La Libertad) sobre subpoblaciones de linfocitos T humanos. Se elabora un extracto crudo y se fracciona por cromatografía de exclusión molecular para aislar las lectinas, las cuales se identifican mediante hemaglutinación y SDS-PAGE. Realiza cultivos de células mononucleares de sangre periférica empleando diferentes concentraciones del extracto y de la fracción seleccionada. En los cultivos con extracto evalúa la proliferación y la activación de linfocitos T por observación microscópica directa y determinación de receptor CD25, respectivamente. En los cultivos con la fracción seleccionada se determina el índice de estimulación, el índice mitótico y se identifican las subpoblaciones de linfocitos T mediante ensayo MTT, obtención de placas metafásicas y citometria de flujo, respectivamente. El extracto crudo y la fracción seleccionada mostraron mayor actividad hemaglutinante sobre glóbulos rojos de conejo comparado con los de carnero y humano. Determina que la fracción contenía una lectina de aproximadamente 43 KDa. Se evidencia proliferación y activación en los cultivos, obteniéndose 43.7±1.4% (p<0.01) de linfocitos T con receptor para IL-2 (CD25+) a 1 µg/mL de extracto. El máximo índice de estimulación fue de 1.22±0.07 a 5 µg/mL, mientras que el índice mitótico fue de 1.5%. El porcentaje de linfocitos CD3+ fue superior respecto al control sin lectinas (p=0.02); mientras que las subpoblaciones CD4+ y CD8+ mantuvieron las mismas proporciones que el control. Se concluye que las lectinas del ecotipo Patón Grande tienen actividad mitogénica sobre linfocitos T y estimulan indistintamente a linfocitos T CD3+/CD4+ y CD3+/CD8+. / Tesis