Automatic annotation of cellular data

Life scientists often need to count cells in microscopy images, which is very tedious and a time consuming task. Henceforth, automatic approaches can be a solution to this problem. Several works have been devised for this issue, but the majority of these approaches degrade their performance in case of cell overlapping. In this dissertation we propose a method to determine the position of macrophages and parasites in uorescence images of Leishmania-infected macrophages. The proposed strategy is mainly based on blob detection, clustering and separation using concave regions of the cells' contour. By carrying out a comparison with other approaches that also addressed this type of images, we concluded that the proposed methodology achieves better performance in the automatic annotation of Leishmania infections.A anotação de células é uma tarefa comum a diversas áreas da investigação biomédica. Normalmente, esta tarefa é realizada de forma manual, sendo um processo demorado, cansativo e propício a erros. Neste trabalho, focamos o nosso interesse na anotação de imagens de uorescência com infeções de Leishmania, que representa um destes casos. Leishmania são parasitas unicelulares que infectam mamíferos, sendo responsáveis por um conjunto de doenças conhecidas por leishmanioses. Leishmania usam vertebrados como hospedeiros residindo dentro dos seus macrófagos. Por conseguinte, um modelo adequado para o estudo destes parasitas é infectar in vitro culturas de macrófagos. A capacidade de sobrevivência/replicação da Leishmania nessas condições arti - ciais pode então ser avaliada por parâmetros, como, por exemplo, a percentagem de macrófagos infectados, o número médio de parasitas por macrófagos infectados e o índice de infeção. Essas métricas são geralmente determinadas pela contagem de parasitas e macrófagos ao microscópio. Ambos os tipos de células podem ser facilmente distinguidos com base no seu tamanho e cor, resultante de diferentes a nidades de corantes uorescentes. A passagem desta tarefa do microscópio para o computador já foi conseguida através de aplicações como o CellNote, contudo, apesar de mais fácil e interativa, a anotação continua a ser manual. A evolução da abordagem manual para um processo automático representa um passo natural e lógico, constituindo o principal objetivo deste trabalho. Para isto iniciámos a investigação pela revisão dos principais métodos de deteção e contagem celular. As características das imagens com infeções de Leishmania impossibilitam a utilização dos métodos estudados, de tal modo que optámos por desenvolver uma nova abordagem, capaz de lidar com as várias especi cidades destas imagens. Também durante o processo de revis ão de literatura analisámos os dois métodos previamente propostos para realizar a anotação automática de infeções de Leishmania. Estes revelaram um desempenho abaixo do requerido pelos parasitologistas, justi cando também o desenvolvimento de uma nova abordagem. Durante a concepção do sistema investigámos diversas técnicas de deteção celular, onde a deteção de blobs se destacou pelos resultados positivos. Para segmentar as regiões citoplasmáticas optámos pela utilização de algoritmos de clustering. Estes não foram capazes de solucionar casos em que existia sobreposição de estruturas celulares, motivando assim o método de separação desenvolvido. Este método baseia-se maioritariamente na análise de contorno, sendo as suas concavidades geradoras de separação entre citoplasmas. Através da combinação destas fases foi possível detetar macrófagos e parasitas com mais precisão. Para con rmar esta conclusão testámos não só a nossa abordagem mas também as duas abordagens previamente desenvolvidas para este problema. Os desempenhos alcançados evidenciam não só uma melhoria comparativamente às restantes abordagens como também mostram que a nossa abordagem assegura resultados satisfatórios comparativamente aos obtidos manualmente. Em suma, o trabalho desenvolvido produziu um sistema capaz de realizar a anotação automática de imagens de uorescência com infeções de Leishmania, tendo originado um artigo aceite para publicação na conferência International Conference on Image Analysis and Recognition (ICIAR) 2013

Microscopic Image Segmentation to Quantification of Leishmania Infection in Macrophages

The determination of infection rate parameter from in vitro macrophages infected by Leishmania amastigotes is fundamental in the study of vaccine candidates and new drugs for the treatment of leishmaniasis. The conventional method that consists in the amastigotes count inside macrophages, normally is done by a trained microscope technician, which is liable to misinterpretation and sampling. The objective of this work is to develop a method for the segmentation of images to enable the automatic calculation of the infection rate by amastigotes. Segmentation is based on mathematical morphology in the context of a computer vision system. The results obtained by computer vision system presents a 95% accuracy in comparison to the conventional method. Therefore, the proposed method can contribute to the speed and accuracy of analysis of infection rate, minimizing errors from the traditional methods, especially in situations where exhaustive repetitions of the procedure are required from the technician.A determinação de parâmetros como taxa de infecção em monocultura de macrófagos cultivados in vitro com Leishmania é fundamental no estudo de candidatos vacinais e novos fármacos para o tratamento de leishmanioses. O método convencional que consiste na contagem de amastigotas no interior de macrófagos, normalmente é realizada por um especialista treinado em microscopia óptica, o que está sujeito a erros de interpretação e amostragem. O objetivo do trabalho é desenvolver um método para a segmentação de imagens como etapa preliminar para o cálculo automático da taxa de infecção por amastigotas. A segmentação é baseada em morfologia matemática no contexto de um sistema de visão computacional. Os resultados obtidos pelo método computacional demonstraram acerto de 95% quando comparados ao método convencional. Conclui-se que a metodologia computacional baseada na segmentação de imagem como pré-requisito para o cálculo de taxa de infecção, pode contribuir para a rapidez e a precisão na obtenção dos resultados e na minimização de erros cometidos no método tradicional, especialmente em situações em que exaustivas repetições do procedimento são exigidas ao observador

Molecular, biochemical and functional analysis of kinesins in Leishmania mexicana

This thesis was previously held under moratorium from 22 August 2019 to 22 August 2021.Leishmaniasis is a vector-borne disease that is caused by several species of the obligate intra-cellular protozoan parasite Leishmania. Leishmaniasis is a tropical and sub-tropical disease affecting between (12 – 15) million people worldwide. An estimated 1.5 to 2 million new cases occur and it causes 70,000 deaths per year. The flagellated protist Leishmania is one of the model organisms to study flagellar assembly. Here, we used L. mexicana as a model to investigate flagellar kinesin motor proteins. Kinesins are a large superfamily (KIFs). More than 15 kinesin families were classified by phylogenetic analysis (Wickstead et al., 2006). Kinesins are motor proteins that convert the energy from ATP hydrolysis into mechanical work to drive cargo along microtubules in a variety of cellular processes, organelle transport and cell division. Disruption of the normal function of these proteins has been shown to lead to many pathologies, including ciliopathy, neurodegenerative diseases and cancers. The current study presents a comprehensive biochemical and cell biological analysis of three kinesins thought to be associated with flagellum formation. Initially the cloning, mapping, and expression of the novel kinesin LmxKin29 were achieved. LmxKin29 is expressed in both the amastigote and promastigote life stages of L. mexicana. LmxKin29 can be assigned to the “orphan” kinesin family. Prior to the beginning of this work, the MAP kinase homologue LmxMPK3 was found to phosphorylate a peptide derived from LmxKin29 encompassing serine 551 and serine 554 (Rosenqvist, 2011; Emmerson, 2014). Here, a full-length GST-fusion protein of wild type LmxKin29 and five different mutants with substitutions of the putative serine or threonine phosphorylation sites, by alanine or aspartate, namely LmxKin29SA, LmxKin29SD, LmxKin29A2, LmxKin29A4 and LmxKin29554A were analysed. Using these mutants, it was possible to narrow down the site that is phosphorylated in activated His-LmxMPK3 as serine 554. To assess the function of LmxKin29 in L. mexicana single and double allele null mutants were generated. Morphological analysis of promastigotes displayed no obvious phenotypic differences comparing the mutants with wild type cells. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Female Balb/c mice infected with ΔLmxKin29-/- null mutant promastigotes did not show a footpad lesion, whereas LmxKin29 add-back clones and single allele knockout clones caused the disease similar to wild type parasites. It was confirmed by ELISA that the serum of mice infected with L. mexicana wild type, single allele mutants and add-back mutants showed increased levels of IgG1 and IgG2a. However, the LmxKin29 null mutant scored very low similar to the level of uninfected mice serving as a negative control. The inability to cause lesions in the infected animal suggests that LmxKin29 is a potential drug target against leishmaniasis. On the other hand the absence of an immune response against the LmxKin29 null mutant clearly rules out these mutant parasites as an attenuated live vaccine. LmxOSM3.1 and LmxOSM3.2 were found to be homologous to flagellar kinesin-2 in trypanosomatids (L. major and T. brucei). They also showed sequence similarity with kinesin-2s of human kinesin KIF3A/B as well as with C. reinhardtii FLA8/FLA10 and C. elegans kinesin-like protein klp-20klp11. LmxOSM3.1GFP localisation was identified along the flagellum of L. mexicana promastigotes, while LmxOSM3.2RFP was localised at the tip of the flagellum. Hence, both kinesins might be involved in flagellum formation. LmxOSM3.1 has been identified previously as a substrate of LmxMPK3 and LmxMPK13. Kinase assays using GST-LmxOSM3.2 with activated LmxMPK3 showed that this kinase cannot phosphorylate LmxOSM3.2 in vitro. We can hypothesise that LmxOSM3.1 and LmxOSM3.2 are core IFT motors that are involved in the assembly and maintenance of the flagellum, whereas LmxOSM3.2 exhibit as an accessory motor that provides flagellum-specific functions.Leishmaniasis is a vector-borne disease that is caused by several species of the obligate intra-cellular protozoan parasite Leishmania. Leishmaniasis is a tropical and sub-tropical disease affecting between (12 – 15) million people worldwide. An estimated 1.5 to 2 million new cases occur and it causes 70,000 deaths per year. The flagellated protist Leishmania is one of the model organisms to study flagellar assembly. Here, we used L. mexicana as a model to investigate flagellar kinesin motor proteins. Kinesins are a large superfamily (KIFs). More than 15 kinesin families were classified by phylogenetic analysis (Wickstead et al., 2006). Kinesins are motor proteins that convert the energy from ATP hydrolysis into mechanical work to drive cargo along microtubules in a variety of cellular processes, organelle transport and cell division. Disruption of the normal function of these proteins has been shown to lead to many pathologies, including ciliopathy, neurodegenerative diseases and cancers. The current study presents a comprehensive biochemical and cell biological analysis of three kinesins thought to be associated with flagellum formation. Initially the cloning, mapping, and expression of the novel kinesin LmxKin29 were achieved. LmxKin29 is expressed in both the amastigote and promastigote life stages of L. mexicana. LmxKin29 can be assigned to the “orphan” kinesin family. Prior to the beginning of this work, the MAP kinase homologue LmxMPK3 was found to phosphorylate a peptide derived from LmxKin29 encompassing serine 551 and serine 554 (Rosenqvist, 2011; Emmerson, 2014). Here, a full-length GST-fusion protein of wild type LmxKin29 and five different mutants with substitutions of the putative serine or threonine phosphorylation sites, by alanine or aspartate, namely LmxKin29SA, LmxKin29SD, LmxKin29A2, LmxKin29A4 and LmxKin29554A were analysed. Using these mutants, it was possible to narrow down the site that is phosphorylated in activated His-LmxMPK3 as serine 554. To assess the function of LmxKin29 in L. mexicana single and double allele null mutants were generated. Morphological analysis of promastigotes displayed no obvious phenotypic differences comparing the mutants with wild type cells. Localisation studies using GFP-tagged LmxKin29 revealed that it is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells LmxKin29 was found at the anterior and posterior ends of the cells. Hence, LmxKin29 might play a role in cytokinesis. Female Balb/c mice infected with ΔLmxKin29-/- null mutant promastigotes did not show a footpad lesion, whereas LmxKin29 add-back clones and single allele knockout clones caused the disease similar to wild type parasites. It was confirmed by ELISA that the serum of mice infected with L. mexicana wild type, single allele mutants and add-back mutants showed increased levels of IgG1 and IgG2a. However, the LmxKin29 null mutant scored very low similar to the level of uninfected mice serving as a negative control. The inability to cause lesions in the infected animal suggests that LmxKin29 is a potential drug target against leishmaniasis. On the other hand the absence of an immune response against the LmxKin29 null mutant clearly rules out these mutant parasites as an attenuated live vaccine. LmxOSM3.1 and LmxOSM3.2 were found to be homologous to flagellar kinesin-2 in trypanosomatids (L. major and T. brucei). They also showed sequence similarity with kinesin-2s of human kinesin KIF3A/B as well as with C. reinhardtii FLA8/FLA10 and C. elegans kinesin-like protein klp-20klp11. LmxOSM3.1GFP localisation was identified along the flagellum of L. mexicana promastigotes, while LmxOSM3.2RFP was localised at the tip of the flagellum. Hence, both kinesins might be involved in flagellum formation. LmxOSM3.1 has been identified previously as a substrate of LmxMPK3 and LmxMPK13. Kinase assays using GST-LmxOSM3.2 with activated LmxMPK3 showed that this kinase cannot phosphorylate LmxOSM3.2 in vitro. We can hypothesise that LmxOSM3.1 and LmxOSM3.2 are core IFT motors that are involved in the assembly and maintenance of the flagellum, whereas LmxOSM3.2 exhibit as an accessory motor that provides flagellum-specific functions

ULK4 and Fused/STK36 interact to mediate assembly of a motile flagellum

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologs have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicates a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localisation throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localise to mammalian motile cilia and we demonstrate here that ULK4 also localises to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/STK36 in a pathway required for stable assembly of motile cilia

Kinetoplastid Genomics and Beyond

This book includes a collection of eight original research articles and three reviews covering a wide range of topics in the field of kinetoplastids. In addition, readers can find a compendium of molecular biology procedures and bioinformatics tools

Molecular analysis of MAP kinase kinase signaling in Leishmania

Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated.Mitogen-activated protein kinase patways play important roles in L. mexicana cell biology. This research characterised two yet unstudied MAP2Ks (LmxPK3 and LmxPK6) in Leishmania and studied putative signal transduction between MAP2Ks and MAPKs involved in regulating flagellum length. LmxPK6 is closely related to the STE7 kinase family, and LmxPK3 is related to CAMK. Recombinant GST-LmxPK6 could not be obtained, but GST-LmxPK3 could be purified in sufficient amounts to prove kinase activity by phosphorylation of the generic substrate MBP.;Only single allele deletion mutants could be generated for LmxPK6. Multiple attempts to obtain a null mutant were unsuccessful. This might suggest that LmxPK6 is an essential kinase of L. mexicana. However, an LmxPK3 null mutant was successfully generated, relying on the LmxMPK12 flanking regions to guarantee sufficient neomycin phosphotransferase resistance marker gene expression. GFP fused to LmxPK3 at either the C-terminus or the N-terminus showed that LmxPK3 localised in the cytosol and flagellum.;A null mutant of LmxPK3 showed similar lesion development in BALB/c mice as wild type L. mexicana, and the lesion-derived amastigotes differentiated back to promastigotes and grew in culture suggesting that LmxPK3 does not play a role in Leishmania differentiation. Hence, LmxPK3 is not a drug target against leishmaniasis. Interactions between LmxPK4 and LmxMPK3 were investigated in vitro by co-expression of the two kinases in Escherichia coli followed by purification and kinase assays.;MS/MS analysis showed that LmxPK4 phosphorylates LmxMPK3 at SER183, THR194 and TYR196 of the TDY motif. Using split-GFP for the first time in Leishmania promastigotes showed an interaction between LmxPK4 and LmxMPK3 in vivo by fluorescence in distinct areas of the cytosol and formation of normal length flagella when expressed in the LmxMPK3 null mutant. A hypothesis of how LmxPK4 and LmxMKK can jointly regulate intraflagellar transport was generated

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition