Doctor of Philosophy / Department of Biology / Jyoti Shah / Phloem-feeding insects like aphids feed on a variety of crop plants and limit plant productivity. In addition they are vectors for important plant viruses. Efforts to enhance plant resistance to aphids have been hampered by lack of sufficient understanding of mechanisms of plant defense against aphids. I have utilized a plant-aphid system consisting of the model plant Arabidopsis thaliana and the generalist aphid, Myzus persicae Sulzer (green peach aphid [GPA]), to study plant response to aphids. These studies have demonstrated an important role of premature leaf senescence in controlling aphid growth in Arabidopsis. Molecular and physiological studies suggest that the Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT 4) gene modulates the GPA feeding-induced senescence process. Furthermore, in comparison to the wild type plants, GPA growth was higher on pad4 mutant plants, suggesting an important role for PAD4 in plant defense against GPA. In contrast, constitutive expression of PAD4 in transgenic Arabidopsis enhanced basal resistance against GPA. Unlike its involvement in plant defense against pathogens, the role of PAD4 in Arabidopsis resistance to GPA is independent of its involvement in phytoalexin biosynthesis and of its interaction with EDS1, a PAD4-interacting protein. Instead, the heightened resistance to GPA in these PAD4 constitutively expressing plants was associated with the rapid activation of leaf senescence. The association of premature leaf senescence in basal defense against GPA is supported by our observation that in comparison to the wild type plant, GPA growth was restricted on the Arabidopsis hypersenescence mutants, ssi2 and cpr5. Gene expression studies suggested some overlap between plant responses to pathogens and aphids, for example, activation of genes associated with the salicylic acid (SA) signaling pathway. However, the characterization of aphid performance on Arabidopsis SA biosynthesis and signaling mutants have ruled out the involvement of SA signaling in controlling aphid growth.

Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process. / Dr. Marianne J. Cronje

Programmed cell death (PCD) is a universal event experienced by all eukaryotes and is an essential process for the maintenance of regular homeostasis. The careful regulation of PCD pathways is beneficial to all organisms and a better understanding of various PCD components and their interactions may enhance overall quality of life and increase longevity of defective organisms. Since a boom of interest was shown towards the study of PCD approximately thirty years prior, a comprehensive understanding of the underlying mechanisms of apoptosis currently exists. Many of the key features involved in PCD have been shown to be conserved across the animal and plant kingdoms and although the overall process of cell death appears to occur in a similar manner in both plants and animals, subtle variations have been identified between these two kingdoms regarding various mechanisms of apoptosis. The major component of cell death in plants and animals appears to be the mitochondria where most of the PCD points of control converge. Heat shock proteins (Hsps) play a vital role in the regulation of PCD and act at an array of points along the PCD pathway. One of the crucial events of PCD is the release of cytochrome c from the mitochondria which proceeds to activate the protease cascade. Hsp70 has been shown to attenuate apoptosis by specifically preventing cytochrome c release into the cytosol of animal cells. A similar correlation between increases of Hsp70 and decreased PCD has been identified in plants, the mechanism of which remains unclear. The objective of this study was to investigate the interaction between increased Hsp70 accumulation and decreased PCD, as indicated by reduced cytochrome c translocation and DNA laddering. The major observations of this study proceeded to show that an increased expression of Hsp70, induced by a mild heat shock, was able to protect tobacco cells against H2O2-mediated cell death. This protection appeared to commence at a mitochondrial level as cytochrome c translocation was clearly inhibited and was confirmed by the absence of downstream DNA laddering. The major findings obtained from this study provided a clearer picture of the mechanisms surrounding the cytoprotective properties of thermotolerant cells, the implications of which are beneficial to post harvest industries, as the ability to postpone PCD provides an advantage enables prolonged shelf lives of various crops. / Dr. Marianne J. Cronjé

Heat shock proteins (HSP), although not considered classical defence proteins, have general cytoprotective properties, which promote survival of cells and organisms. Hsp70, in particular, provides resistance to the harmful consequences of various forms of otherwise damaging or even lethal stress including pathogen infection. Increased levels of Hsp70, due to stable transfection of cells with hsp70 genes, or elevated expression in response to stress, generally correlate with the hindrance of cell death processes triggered by a variety of noxious stimuli or toxic agents. The effect of lipopolysaccharides (LPS), the major constituent of the outer membrane of the cell wall (envelope) of almost all Gram-negative bacteria, on Hsp70/Hsc70 expression in plants is unknown. In various mammalian systems, LPS has been shown to induce Hsp70 accumulation, along with programmed (apoptotic) cell death. Contrary to the effects of LPS on animal hosts however, LPS does not elicit cell death in plants, but rather pre-treatment with LPS fraction can prevent or delay the so-called hypersensitive response (HR), thus sensitizing plant tissue to respond more rapidly, or to a greater extent, to subsequently inoculated phytopathogenic bacteria. Elevated levels of reactive oxygen species (ROS) reportedly contribute to stress sensing and hsp gene activation, and subsequent Hsp70 induction, during the stress response. Increased ROS production can also trigger cell death via either programmed cell death (PCD), an active (i.e., energy-dependent) physiological suicide mechanism that is genetically regulated, or uncontrolled necrosis, an accidental, lytic form of cell destruction passively triggered by severe trauma or injury. In plants specifically, ROS may be involved in PCD activation during the HR. As a pathogen-associated molecular pattern (PAMP) or general elicitor of defence or resistance-related responses, LPS may trigger some defence-related responses, including an oxidative burst (manifest as increased levels of reactive oxygen species or ROS) in certain plant cells as it does in animal systems. However, there is conflicting evidence that shows that LPS treatment does not elicit an oxidative burst in plants. The aim of this study was to determine the effect of LPS isolated from Ralstonia solanacearum, an extremely harmful soil-borne bacterium that causes Southern wilt in over 200 plant species by infecting the host’s roots and invading the xylem vessels, on Hsp70/Hsc70 accumulation, cell death and ROS production in suspension-cultured tobacco (Nicotiana tabacum) cells, in order to gain a better understanding of the inter-relationship between these three phenomena in plant cells responding to LPS(Ralstonia). Western (immuno)blot analysis was used to study the unknown effect of LPS(Ralstonia) on Hsp70/Hsc70 accumulation in tobacco cell suspensions. LPS(Ralstonia) (all concentrations and time periods studied) generally suppressed Hsp70/Hsc70 accumulation. However, only exposure to 100 μg LPS/ml for 3 h caused a significant reduction (P < 0.05). Therefore, there was an early suppression of Hsp70/Hsc70 accumulation in response to 100 μg LPS(Ralstonia)/ml. To determine whether the observed LPS-mediated attenuation in Hsp70/Hsc70 accumulation was due to an increase in cell death in these cells, we investigated the effect of LPS(Ralstonia) on i) the general viability of the cells, and ii) the integrity of nuclear or genomic DNA extracted from LPS-treated suspension-cultured tobacco cells. The AlamarBlue™ (AB) assay was used to investigate the general cell viability in response to LPS(Ralstonia) treatment. LPS(Ralstonia) (all concentrations and time intervals studied) did not significantly affect the overall viability of the cells. Because treatment of tobacco cell suspensions with LPS(Ralstonia) did not result in a significant decrease (P < 0.05) in AB reduction, it was presumed that LPS(Ralstonia) did not appreciably compromise metabolic activity and was therefore not particularly toxic to these cells. Genomic DNA from cells undergoing PCD-associated internucleosomal DNA fragmentation (IDF) typically runs as a ladder of internucleosomal-sized DNA fragments corresponding to multimers of ca. 180 bp in agarose gels. In contrast, random DNA cleavage, usually manifest as smearing of nuclear DNA following agarose gel electrophoresis, is a token of uncontrolled necrosis. Therefore, if so-called “DNA laddering” is observed following agarose gel electrophoresis of genomic DNA extracted from suspension-cultured tobacco cells exposed to LPS(Ralstonia) then it can be assumed that LPS(Ralstonia) induced PCD. Alternatively, if a long, continuous “necrotic smear” is evident after electrophoretic separation of nuclear DNA from LPS-treated cells then LPS(Ralstonia) clearly induced uncontrolled necrosis. Whether or not LPS(Ralstonia) induced PCD-associated IDF or necrotic smearing was determined by investigating genomic DNA fragmentation (or DNA integrity) in response to LPS(Ralstonia) iii treatment. Although no typical DNA ladders were detected following electrophoresis of DNA isolated from LPS-treated cells, PCD may still have transpired. However, this is highly unlikely. No necrotic smearing was evident in LPS-treated samples either, which verifies the hypothesis that LPS(Ralstonia) (25–100 μg/ml) did not induce uncontrolled necrosis in suspension-cultured tobacco cells. In fact, these concentrations of LPS(Ralstonia) did not seem to significantly compromise DNA integrity given that LPS(Ralstonia) (25–100 μg/ml) generally had no appreciable effect on genomic DNA fragmentation (compared to untreated control samples). Incidentally, 24-h exposure of tobacco cell suspensions to higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) may have resulted in partial DNA cleavage and/or degradation. Exposure of tobacco cell suspensions to 400 μg LPS(Burkholderia)/ml for 7 days may also have evoked partial DNA cleavage and/or degradation. Whether this cleavage and/or degradation occurred deliberately by means of a fixed or predetermined mechanism or randomly by an uncontrolled mechanism remains uncertain. Finally, the H2DCF-DA (2′, 7′-dihydrodichlorofluorescein-diacetate) fluorescence assay was used to investigate the effect of LPS(Ralstonia) on ROS production, a common factor in the regulation of HSP expression and cell death activation. LPS(Ralstonia) treatment (25–100 μg/ml) generally increased ROS levels in suspension-cultured tobacco cells (compared to untreated control cells). Exposure to 75 μg LPS(Ralstonia)/ml resulted in a particularly prominent elevation in ROS levels almost instantaneously. Incidentally, higher concentrations of LPS(Ralstonia) (500 and 1000 μg/ml) resulted in decreased ROS levels at some point during the assay. Although LPS(Ralstonia) (100 μg/ml for 3 h) significantly decreased Hsp70/Hsc70 accumulation in tobacco cell suspensions, cell death did not appear to be induced. In fact, LPS(Ralstonia) had no effect on general cell viability and appeared to be ineffective at causing PCD-associated IDF (DNA laddering) or necrotic smearing regardless of concentration or time of exposure. Despite these findings, treatment of suspension-cultured tobacco cells with LPS(Ralstonia) (≤ 100 μg/ml) resulted in a mild increase in ROS production. Although the exact mechanism(s) by which LPS(Ralstonia) suppressed Hsp70/Hsc70 accumulation is elusive, our results suggest that the suppression is not related to excessive LPS-mediated injury caused by excessively high ROS levels or increased cell death. We speculate that the prevention of HR-related PCD often observed in plants that are pre-treated with LPS and subsequently inoculated with phytopathogenic bacteria may be dependent on the LPS-mediated suppression of cytosolic Hsp70 expression. / Dr. M.J. Cronje

M.Sc. / Established therapies currently in use for the treatment of breast cancer are high risk, since their employment harbors multiple undesirable and detrimental side effects. In many cases these are associated with poor therapeutic outcome managing only to briefly extend patient lifespan. As a result, many newly designed treatments have surfaced that aim to effectively remove cancerous tissue and improve survival rates while minimizing the aggressive onsets to the patient. Photodynamic Therapy (PDT) has, for a long time, been targeted as a combatant for cancer. Its therapeutic mechanism is based on the tumor-specific intracellular localization of a photosynthetic compound, i.e: photosensitizer, prior to its irradiation-mediated excitation thereby generating high levels of reactive oxygen species (ROS). At the molecular level, this causes irreversible photodamage to vital intracellular targets resulting in cell death. The plasma membrane-, mitochondrial-, lysosomal-, and endoplasmic reticulum systems are all prime targets of PDT and vary in susceptibility depending on both the type of cancer being treated and the photosensitizer administered. Newly designed photosensitizers are governed by their enhanced structural properties to localize and therefore target certain areas of a cell. Since each cancer type has a unique set of susceptible and resistant characteristics, knowledge of each new photosensitizers’ range, efficiency, and mechanism of cell death is required. This enables pairing of these drugs to appropriate cancer types for maximal PDT effect. Here, two newly designed metallo-phthalocyanine photosensitizers, AlPcSmix and GePcSmix, were analyzed for their photodynamic effect on the estrogen-positive, breast cancer cell line, MCF-7. Being one of the most reliable cell lines, it is a prominent research model because it mimics the problems encountered with tumor resistance to therapy induced cell death via pathway restrictions. Photosensitizer administration and excitation by light irradiation to this cell culture system was therefore referred to as in vitro Photodynamic Treatment (in vitro PDT) and not Therapy. ii Initial dosage and time responsive studies confirmed that 35 μM AlPcSmix and 115 μM GePcSmix both excited using 15 J/cm2 at 680 nm proved most effective in reducing viability, whilst individually contributing little adverse influence to cellular homeostasis. Using these dosages, in vitro PDT analysis on several cellular parameters indicated a complex mode of cell death was induced. Morphology revealed typical markers consistent with apoptotic, autophagic and necrotic cell death while variations in proliferation and cytotoxicity levels were inconsistent with stress responses observed. This correlated with the detection for four common apoptotic markers which also revealed discrepancies within the death pathway as possible mutational deficiencies may have rendered MCF-7 cellular systems incomplete. Taken together, the wide range of cellular parameters studied suggests cells undergo a mixture of death modes interchanging via a complex system of molecular switches over time that concludes in secondary necrosis. This was attributed to the assortment of sulphonated phthalocyanine species enabling a broad intracellular distribution range coupled with the non-specific targeting action typical of the ROS generated, in addition to the absence of a phagocytic conclusion to the death process. In vitro PDT with AlPcSmix was shown to harbor a greater toxicity to GePcSmix as cells completed the death response within a shorter time period however, both promoted MCF-7 population cell death sufficient enough to warrant further study for their use as potential agents in the PDT of breast cancer.

Background: Heat-shock (HS) proteins (HSP) are induced or increasingly expressed to protect against lethal environmental stresses. Hsp70 in particular, provides protection against various stresses including oxidative stress, is implicated in thermotolerance and appears to have an anti-apoptotic function. Anti-inflammatory salicylates potentiate the induction of the 70 kDa HSP (Hsp70) in mammals in response to HS, enhance thermotolerance and induce apoptosis. In plants, salicylic acid (SA) is a natural signalling molecule, mediating resistance in response to avirulent pathogens. The effects of salicylic acid-mediated increases in Hsp70/Hsc70 expression and its relation to events associated with PCD/ apoptosis in plants are unknown. Hypothesis and Objectives: The hypothesis studied in this investigation was that SA influences Hsp70 expression similar to that found in mammalian cells and may influence the choice between survival or death, whether apoptosis or necrosis. In order to verify this hypothesis the effect of SA alone or in combination with HS on Hsp70/Hsc70 accumulation and events associated with apoptosis were investigated through three main objectives: 1) Determine whether SA in plants, as in mammalian cells, can potentiate heat-induced Hsp70/Hsc70 accumulation or induce Hsp70/Hsc70 by itself at elevated levels. This was done by investigating the effect of SA at various concentrations on Hsp70/Hsc70 expression at normal temperatures or following heat. 2) Establish flow cytometry as a rapid and quantitative alternative for the evaluation of Hsp70 accumulation in plant protoplasts to be evaluated in concert with various parameters indicative of cellular integrity. 3) Investigate whether Hsp70/Hsc70 expression modulated by SA influences cell death (apoptosis/necrosis) or associated events such as mitochondrial membrane permeability (MMP) or reactive oxygen species (ROS) in plant protoplasts using flow cytometry. Materials and Methods: The effect of SA alone or in combination with HS on Hsp70/Hsc70 levels in tomato cells was investigated using biometabolic labelling and Western blotting. A flow cytometric assay was developed to determine Hsp70/Hsc70 levels in tobacco protoplasts. MMP and ROS were monitored by the fluorescent probes DiIC1(5) and H2DCFDA respectively, phosphatidylserine externalisation by annexin V binding and DNA fragmentation by the TUNEL assay in protoplasts treated with SA and/or HS. Results: Results obtained in the attainment of the three main objectives were: 1) In plants, as in mammals, low concentrations of SA do not induce Hsp70/Hsc70 but significantly potentiate heat-induced Hsp70/Hsc70 levels while cytotoxic levels significantly induce Hsp70/Hsc70. In cell suspension cultures, this induction was preceded by increased membrane permeability. 2) Flow cytometry can be implemented as a rapid, quantitative alternative to detect intracellular Hsp70/Hsc70 accumulation in protoplasts. 3) In protoplasts exposed to low doses of SA at normal temperatures, PCD/apoptosis is increased as reflected by increased DNA fragmentation and phosphatidylserine externalisation, but not by increased MMP or ROS. High doses of SA were associated with increased levels of necrosis. Exposure of protoplasts to low doses of SA in combination with HS showed suppression of PCD/apoptosis (reflected by decreased DNA fragmentation and phosphatidylserine externalisation), accompanied by decreased levels of ROS and increased MMP. Discussion: These results suggest that SA-mediated increases in Hsp70/Hsc70 accumulation at normal temperatures are associated with cellular damage and protect cells against necrosis. On the other hand, low doses of SA that potentiate heat-induced Hsp70/Hsc70 accumulation abrogated the induction of apoptosis that was induced by low doses of SA at normal temperatures. The anti-apoptotic effects of Hsp70 could therefore influence plant resistance by interfering with the execution of PCD. These results could contribute to our understanding of heat-induced disease susceptibility, and the manipulation of SA-modulated Hsp70/Hsc70 should be carefully considered in the light of its ability to affect cell death, which may be advantageous or deleterious to the plant cell. / Prof. L. Bornman

The precise structure of the adult Drosophila eye results from a coordinated process of cell sorting, differentiation and selective cell death in the retinal epithelium. Mutations in the gene echinus cause supernumerary pigment cells due to insufficient cell death. This study reports the identification of echinus and the characterization of its role in Drosophila retinal development. Using a combination of deletion mapping, gene expression analysis and genomic sequencing, echinus was cloned and several alleles were sequenced. echinus encodes a ~180kDa protein containing an ubiquitin hydrolase domain at its N-terminus and a polyglutamine tract at its C-terminus. echinus is expressed in the retina during pupal development and mutants of echinus have decreased levels of apoptosis during several stages of retinal development. Defects in the cell sorting process that precedes cell death are also observed in echinus loss-of-function mutants and echinus overexpression can cause defects in ommatidial rotation and the morphology of cone cells. echinus is a positive regulator of DE-cadherin and Enabled accumulation in adherens junctions of retinal epithelial cells. Genetic interactions were observed between echinus and the genes wingless, enabled and expanded. An immunofluorescence assay in Drosophila S2 cell cultured demonstrated that Echinus localizes to intracellular vesicles that do not appear to be endocytic in nature, and the C-terminal region of Echinus was shown to be necessary for this association. A protein interaction screen using an immunoprecipitation and mass spectrometry approach identified interactions between Echinus and the vesicle coat protein Clathrin, the scaffolding protein RACK1 and the casein kinase I epsilon (Dco). Co-immunoprecipitation additionally identified an interaction between Echinus and Enabled. This work has revealed echinus to be an important regulator of cell sorting and adherens junction formation in the developing retina and has identified multiple interactions between echinus and enabled, a regulator of the actin cytoskeleton. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

No description available.

Cell fate decisions incorporate distinct and overlapping mechanisms. The activity of caspase 3 was initially understood to be a cell death restricted event, however numerous studies have implicated this enzyme in the regulation of both differentiation and proliferation. How the activity of caspase 3 promotes a non-death cell fate remains unclear. Here we examine the role caspase 3 activity plays during skeletal muscle differentiation; in particular we explore the hypothesis that the mechanism of inducing DNA strand breaks during cell death is also a key feature of differentiation, albeit with a distinctly different outcome. We delineate the transient formation of Caspase 3/Caspase activated DNase (CAD) dependent DNA strand breaks during differentiation. The formation of these breaks is essential for differentiation and the regulation of specific genes. In particular expression of the cell cycle inhibitor p21 is related to the formation of a DNA strand break within the gene’s promoter element. Further, we explored the genome wide association of CAD using Chromatin Immunoprecipitation coupled to high through put sequencing (ChIP-seq). This approach identified a potential role for Caspase3/CAD in regulating the expression of Pax7. Here, a CAD directed DNA strand break in the Pax7 gene is correlated with decreased Pax7 expression, an outcome that has been shown to be critical for progress of the myogenic differentiation program. The regulation of Pax7 expression through a CAD induced DNA strand break raises an intriguing connection between this regulation and oncogenic transformation observed in alveolar rhabdomyosarcoma. The putative site of CAD induced DNA strand breaks that promote decreased Pax7 expression during differentiation corresponds to site of chromosomal translocations responsible for Pax7 fusion events in alveolar rhabdomyosarcoma.

Orientadores: Maria Ligia Rodrigues Macedo, Tomomasa Yano / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T00:07:13Z (GMT). No. of bitstreams: 1 Ventura_ClaudioAngelo_D.pdf: 5407567 bytes, checksum: 8408eb98403ba9f693c16020c7c85169 (MD5) Previous issue date: 2006 / Resumo: Lectinas constituem uma classe de proteínas ou glicoproteínas que são capazes de ligar-se, reversivelmente e seletivamente, a carboidratos sem causar transformação química. Estudos têm mostrado que a ligação de lectinas a carboidratos de superficie celular de células normais e malignas leva a vários efeitos biológicos tais como proliferação celular e apoptose. Neste trabalho, nós investigamos os efeitos citotóxicos induzidos por TEL (uma lectina, isolada de sementes de Talisia esculenta, a qual preferencialmente liga-se a resíduos de manose) sobre linhagens de células tumorais e não-tumorais. Células Vero (rim de macaco verde afiicano) e Rela (carcinoma cervical humano) tratadas com TEL exibiram perda da integridade da membrana plasmática, retração celular, condensação da cromatina, fragmentação nuclear e formação de corpos apoptóticos. Além disso, células Vero e Rela tratadas com TEL também revelaram uma drástica desorganização do citoesqueleto de actina. A Fragmentação intemuc1eossomal do DNA foi detectada pelo método de TUNEL e eletroforese em gel de agarose. Os efeitos citotóxicos induzidos por TEL foram progressivos e mostraram que as alterações morfológicas e os danos ao DNA ocorreram após a perda da integridade da membrana. Adicionalmente, TEL inibiu significantemente a proliferação das células Rela de maneira dependente da concentração. Nós também mostramos através de microscopia de tluorescência que TEL é intemalizada nas células Vero dentro de pequenas vesículas que depois se acumulam da região perinuclear; nas células Rela, a intemalização não foi observada. Foi estabelecido que a atividade das caspases-3 e -9 aumentaram nas células Vero e Rela de uma maneira dependente do tempo. Em contraste com as células Vero, a atividade da caspase-3 precedeu a ativação da caspase9 nas células Rela. Os efeitos citotóxicos e a intemalização de TEL foram bloqueados pela mano se, sugerindo que a ligação de TEL ao carboidrato específico na superficie celular é um pré-requisito para esses processos. Os resultados mostraram que a morte celular induzida por TEL nas células Vero e Rela seguiu uma série de eventos que culminou em um modo apoptótico de morte celular. Finalmente, uma vez que as células Rela foram altamente sensíveis a citotoxicidade induzida pela lectina, TEL merece futuras investigações porque suas propriedades podem ser uma ferramenta útil na terapia contra câncer cervical humano / Abstract: Lectins constitute a class of proteins or glycoproteins which are capable of binding carbohydrates selectively and reversibly without causing their chemical transformation. Studies have shown that lectin binding to cell surface carbohydrates of normal and malignants cells triggers various biological effects such as cellular proliferation and apoptosis. In this work, we investigate the cytotoxic effects induced by TEL (a lectin, isolated from Talisia esculenta seeds, which binds preferentially to mannose residues), on tumoral and non-tumoral cell lines. TEL- treated Vero (African green monkey kidney) and Hela (human cervical carcinoma) cells exhibited loss of plasma membrane integrity, cellular retraction, chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. Furthermore, TEL-treated Vero and Hela cells also revealed a drastic disorganization of the actin cytoeskeleton. Internucleossomal DNA fragmentation was detected by TUNEL method. and agarose gel electrophoresis. TEL-induced cytotoxic effects were progressive and showed that the morphological alterations and DNA damage occurred after the loss of membrane integrity. Additionally, TEL significantly inhibited the proliferation of Hela cells in a concentration- dependent manner. We also show through of fluorescence microspy that TEL is internalized in Vero cells into small visicles that further coalesce in the perinuclear region; in Hela cells, TEL internalization was not observed. It was established that caspase-3 and -9 activity increased in Vero and Hela cells after TEL treatment in a time- dependent manner. In contrast to Vero cells, caspase- 3 activity preceded the activation of caspase-9 in Hela cells. The cytotoxic effects and the internalization of TEL was blocked by manose, suggesting that the binding of TELto specific carbohydrate on the cell-surface is a prerequisite for these processes. The results showed that TEL- induced cell death in Vero and HeLa cells followed by down stream events leading to apoptotic mode of cell death. Finally, since Hela cells were highly sensitive to lectin-induced cytotoxicity, TEL merits further investigation due to its properties can be a useful tool in therapy against human cervical cancer / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular