**3.1. Basic and upper secondary education**

New national-level curricula for basic (primary and lower secondary) and upper secondary education were prepared between 2012 and 2014 in close collaboration with teachers, teacher educators and providers of education (municipalities) [26, 27]. Both curricula emphasise the learning of twenty-first century competences and offer support to teachers as they confront such key questions as: what will education mean in the future, how can education prepare all young people for the future, what competences will be needed in everyday and working life and what kinds of learning environments and practices or teaching methods would best produce the desired education and learning outcomes.


**1. Introduction**

Italy is the second country in Europe for manufacturing production and the nation week in the world from the point of view of industrialization, albeit with a limited territory and population. For many decades, this process of industrialization has had to face various problems relating to situations of risk of accidents at work. Each economic phase involves the definition of different risk conditions. The passage from the industrial model to a tertiary economy and to the "Fourth capitalism" has led many countries to the overcoming of the central function of manufacturing production, for the promotion of a system based on services. This has also happened to countries with a great industrial tradition, such as Italy and Germany. However, this situation has not entirely limited the conditions of exposure to the risk of accidents at work: the maintenance of a significant presence in industrial production has been accompanied by the new risk conditions that characterize the new economic models and which extend to the sector of tertiary and services. For this reason, in the last 20 years, Italian legislation and the system of employment contracts have placed a strong focus on the prevention of risks at work, introducing important obligations for each company and specific figures, such as the Safety Manager. Italy has a very significant law regarding the prevention and protection of health and safety in the workplace, the "Consolidated text for health and safety in the workplace" [1] (Legislative Decree no. 81 of 2008), a national body called Inail which carries out insurance activities, but also for the prevention of occupational risks, information, training, and assistance in the field of safety and health at work and a national inspection agency for checks in companies. However, the issue of

exposure to the risk of accidents at work remains a very present phenomenon, which must be considered due to the following in-depth elements, which constitute important aspects of the evolution of the economic and production system, destined to produce effects also in the coming years:

This situation must be seen as a whole and determines significant impacts on society and on the health of citizens, which is useful to examine and evaluate.


**2.1 Agency theory**

Agency theory was introduced into management literature by Jensen and Meckling (1976). The theme is based on economic theory and it describes a principal-agent relationship between owners (such as stockholders) and executives, with top executives acting as agents whose personal interests do not naturally align with shareholder interests.

The principal-agent relationship involves a transfer of trust and duty to the agent while assuming that the agent is opportunistic and will pursue interests, including executive fraud, which are in conflict with those of the principal. This potential conflict of interests is often referred to as "the agency problem" (Davis et al. 1997). A typical solution to the agency problem is to structure executive incentives, such as stock options, in such ways that they align executive behavior with stockholder goals. Another common solution to the agency problem is for the board of directors to control and curtail the "opportunistic behavior" of the executives by, for example, the audit committee (Donaldson and Davis 1991).

However, the studies cited in Davis et al. (1997) indicate corporate executives are extremely complex human beings and the agency problem persists. Recent studies by Daily et al. (2003) and Sundaramurthy and Lewis (2003) also show that, in practice, corporate executives have power to counteract the board's control over them. For example, the corporate executives can exercise influence over the board because they are more in tune with daily operations of the company, or they exercise influence over succession of the board to ensure that board members who agree with them are appointed. Finally, Bebchuk and Fried (2004) argue that corporate executives' influence over the board of directors on pay setting can explain a wide range of compensation practices and patterns. This includes ones that have long been viewed as puzzles by economists such as why pay is higher and less sensitive to bad performance, including fraud, in corporations in which executives are more entrenched or have more power vis-à-vis the board.


Naturally, changes in resource supply also have important effects [75]. Increasing the carrying capacity of a resource that one species depends on directly increases that species' *κ* value, and so can increase its ability to compete with another species that does not depend on that resource. This other species would thus be disadvantaged, potentially driving it extinct. However, not just average fitness, *κ*, but niche overlap, *ρ*, would be affected by the increase in supply of a particular resource because *ρ* depends on resource carrying capacities (see Table 1). In particular, *ρ* would decrease with an increase in the carrying capacity of a resource that only one species uses. Nevertheless, the change in the *κ* value has the larger effect confirming the conclusion that changes directly benefiting only one species will harm its competitors indirectly. Effects like this are most clearly seen for invasive species where an increase in resource supply, for example nitrogen deposition for an invasive plant species [76, 77], gives advantages to invaders, depressing native species. However, these concerns apply not only between invaders and native species but between native species where factors helping just some species may well negatively affect others in the same guild [75].

These various effects stemming from changes in resource richness have their counterparts in habitat availabilities because habitat variation in space can have effects similar to resource diversity, as explained above in the section on variation in time and space. Thus, increasing the availability of a habitat type specific to one species, can negatively affect another species that does not use that habitat type, but shares other habitat types with the species that does.

beyond MacArthur's [27] beginnings. Moreover, Shigesada [69] provides an explicit

The fact that different species do differ from one another ecologically means that changes in the environment do affect different species differently or to different degrees. Under the individual-species approach to conservation and management, this would simply mean that different species are at risk to different degrees from some change in the environment due to human activities. Under the interactive model given here, changes in the environment need not directly affect a species for it to be affected indirectly through its linkages with other species. The fitness measures *κ* reveal this most simply. A mortality rate of a particular species might be changed by hunting, elimination of a predator, or changes in physical stress in the environment. That change would have a direct effect on that species, either increasing or decreasing its abundance and perhaps putting it at risk, but that change would also have effects on a species' relationships with others. In particular, in the MacArthur model, we see that the ratio of the *κ* values would be affected. An increase in the *κ* ratio in

One example of this *κ*-ratio analysis is its application to understanding invasive species of large effect [30, 70]. A large *κ* value in a particular system would allow a species to invade that system, but would have also have the effect of depressing native species and potentially driving them extinct. There are a number of ways that an invasive species might achieve a larger *κ* value including low susceptibility to natural enemies in the invaded system [71], advantages in resource harvesting [72], and lower sensitivity to competition than the native species [73]. Alternatively, an invasive species might have a larger *κ* value than native species because habitat degradation has increased stresses on native species, lowering their *κ* values, and rendering the system vulnerable to invasion [30, 74]. Viewed from another perspective, a native species might become endangered not because some environmental change has directly affected it, but because other guild members have had their *κ* values

Naturally, changes in resource supply also have important effects [75]. Increasing the carrying capacity of a resource that one species depends on directly increases that species' *κ* value, and so can increase its ability to compete with another species that does not depend on that resource. This other species would thus be disadvantaged, potentially driving it extinct. However, not just average fitness, *κ*, but niche overlap, *ρ*, would be affected by the increase in supply of a particular resource because *ρ* depends on resource carrying capacities (see Table 1). In particular, *ρ* would decrease with an increase in the carrying capacity of a resource that only one species uses. Nevertheless, the change in the *κ* value has the larger effect confirming the conclusion that changes directly benefiting only one species will harm its competitors indirectly. Effects like this are most clearly seen for invasive species where an increase in resource supply, for example nitrogen deposition for an invasive plant species [76, 77], gives advantages to invaders, depressing native species. However, these concerns apply not only between invaders and native species but between native species where factors helping just some species may well negatively affect others in the same guild [75]. These various effects stemming from changes in resource richness have their counterparts in habitat availabilities because habitat variation in space can have effects similar to resource diversity, as explained above in the section on variation in time and space. Thus, increasing the availability of a habitat type specific to one species, can negatively affect another species that does not use that habitat type, but shares other habitat types with the species that does.

representation of spatial competition models in the MacArthur framework.

one species favor disadvantages another, potentially leading to its extinction.

elevated by changes that directly benefited them.

This outcome can be understood intuitively: one species increases in abundance when a valuable habitat type becomes more available, and, due to dispersal between habitat types, its abundance everywhere is increased. It thus has greater competitive effects on other species in all habitats. From the perspective of the theory above, average fitnesses and niche overlap change in the same way as when resource supply is changed. Similar affects apply to temporal environmental variation, an issue of particular concern with climate change [78]. For example, desert annual plants are believed to coexist with one another because different species are favored in different years, depending on the weather [19, 55]. Changes in weather patterns can change the relative *κ* values of the species if the weather becomes on average more favorable for some than for others. Changes in disturbance regimes can have similar effects through changes in the patterns of spatio-temporal variation [79].

Although systems where coexistence relies on disturbance are sometimes regarded as nonequilibrial [80], they do not escape these general priniciples. Individual species may be adapted to survive and even take advantage of temporal change, but there is every reason to expect that individual species, and the system as a whole, are sensitive to changes in the average frequency and intensity of environmental events, such as disturbances [81, 82]. Surviving and taking advantage of temporal change may rely on life-history attributes such as dormancy, but may also depend on dispersal to and from refuge habitats that escape change either permanently or at given time (e.g when a fire sweeps through) [83]. Maintaining landscape connectivity, for example through maintenance of dispersal corridors, is especially important in such circumstances [84].

Finally, we come to the important question of trophic structure. Recognition that predation and competition can have similar roles in diversity maintenance, and that the effects that each has can be undermined by the other, depending on their relative strengths, means that maintenance of trophic structure is vital for conservation [85]. There is much concern about loss of large predators in many systems [86-89]. These predators are often valued for their own sake. Conservation measures focused on them might aim merely for their persistence in nature, rather than for maintenance of the roles that they have in systems [90, 91]. For example, practices in the United States relating to wolf restoration face contentious arguments over the mere persistence of wolves, versus maintaining their numbers so that the ecosystem roles are restored as well [92, 93]. Discussion of trophic cascades, which are increasingly seen in marine systems, as the larger species are eliminated, focus much on the overall abundance of a given trophic level [94, 95]. However, the theory discussed here emphasizes that decimating one trophic level will affect the diversity maintenance roles that it has in other trophic levels for which is either a predator trophic level or a resource trophic level, as has been found empirically in some systems [96].

#### **4. Conclusions**

Taken together, these principles and examples reemphasize that species should not be managed in isolation. Factors improving the situation for one set of species may degrade it for others. Maintaining trophic structure, physical environmental structure, disturbance regimes, and spatial connectivity are all common sense ideas that are backed up by the theory of diversity maintenance mechanisms.

#### **5. Acknowledgements**

This research was supported by NSF grant DEB-0542991.

[21] A. M. Siepielski and M. A. McPeek, "On the evidence for species coexistence: a critique

[22] A. L. W. Sears and P. Chesson, "New methods for quantifying the spatial storage effect: an illustration with desert annuals," *Ecology,* vol. 88, pp. 2240-2247, 2007. [23] J. A. Endler, *Natural Selection in the Wild*. Princeton, New Jersey: Princeton University

[24] E. D. Brodie, A. J. Moore, and F. J. Janzen, "Visualizing and quantifying natural selection," *Trends in Ecology and Evolution,* vol. 10, pp. 313-318, 1995. [25] E. L. Charnov, Life History Invariants: some explorations of symmetry in evolutionary

[26] P. B. Adler, J. HilleRisLambers, and J. M. Levine, "A niche for neutrality," *Ecology*

[27] R. MacArthur, "Species packing and competitive equilibrium for many species,"

[28] P. Chesson, "MacArthur's consumer-resource model.," *Theoretical Population Biology,*

[29] R. Haygood, "Coexistence in MacArthur-style consumer–resource models," *Theoretical*

[30] K. Shea and P. Chesson, "Community ecology theory as a framework for biological invasions," *Trends in Ecology and Evolution,* vol. 17, pp. 170-176, 2002. [31] P. L. Chesson and R. R. Warner, "Environmental variability promotes coexistence in lottery competitive systems," *The American Naturalist,* vol. 117, pp. 923-943, 1981. [32] P. Chesson, "Multispecies competition in variable environments," *Theoretical Population*

[33] P. Chesson and M. Rees, "Commentary on Clark et al . ( 2 0 0 7 ): Resolving the

[34] D. W. Purves and S. W. Pacala, "Ecological drift in niche-structured communities:

[35] P. Amarasekare, "Interference competition and species coexistence," *Proceedings of the Royal Society of London Series B-Biological Sciences,* vol. 269, pp. 2541-2550, 2002. [36] R. M. Callaway, Positive Interactions and Interdependence in Plant Communities.

[37] R. D. Holt and G. A. Polis, "A theoretical framework for intraguild predation," *The*

[38] R. D. Holt, "Spatial heterogeneity, indirect interactions, and the coexistence of prey

[39] R. T. Paine, "Food web complexity and species diversity," *American Naturalist,* vol. 100,

[40] J. Lubchenco, "Plant species diversity in a marine intertidal community: importance of

[41] R. D. Holt, "Predation, apparent competition, and the structure of prey communities,"

herbivore food preference and algal competitive abilities," *American Naturalist,* vol.

neutral pattern does not imply neutral process," in *Biotic Interactions in the Tropics*, B. D., M. Pinard, and S. Hartley, Eds. Cambridge, UK: Cambridge University Press,

biodiversity paradox," *Ecology Letters,* vol. 10, pp. 659-661, 2007.

of the coexistence program," *Ecology,* vol. 91, pp. 3153-3164, 2010.

Fundacion BBVA, 2008, pp. 119-164.

*Letters,* vol. 10, pp. 95-104, 2007.

*Biology,* vol. 45, pp. 227-276, 1994.

vol. 37, pp. 26-38, 1990.

2005, pp. 103-138.

pp. 65-75, 1966.

112, pp. 23-39, 1978.

Dordrecht :: Springer,, 2007.

*American Naturalist,* vol. 149, pp. 745-764, 1997.

species," *American Naturalist,* vol. 124, pp. 377-406, 1984.

*Theoretical Population Biology,* vol. 12, pp. 197-229, 1977.

ecology. Oxford: Oxford University Press, 1993.

*Theoretical Population Biology,* vol. 1, pp. 1-11, 1970.

*Populaiton Biology,* vol. 61, pp. 215-223, 2002.

Press, 1986.

Camacho, A. Elosegui, C. Gracia, M. Estrada, J. C. Senar, and J. M. Gili, Eds. Bilbao:

#### **6. References**

[1] M. L. Rosenzweig, *Species Diversity in Space and Time*. Cambridge: Cambridge University

[2] J. Roughgarden, "Community structure and assembly," in *Perspectives in Ecological*

[3] J. M. Chase and M. A. Leibold, *Ecological Niches: linking classical and contemporary*

[4] S. A. Levin, "Community equilibria and stability, and an extension of the competitive exclusion principle," *American Naturalist,* vol. 104, pp. 413-423, 1970. [5] P. Chesson, "Mechanisms of maintenance of species diversity," *Annual Review of Ecology*

[6] [P. Chesson and N. Huntly, "The roles of harsh and fluctuating conditions in the dynamics of ecological communities," *The American Naturalist,* vol. 150, pp. 519-553, 1997. [7] S. P. Hubbell, *The unified neutral theory of biodiversity and biogeography*. Princeton, NJ:

[8] I. Volkov, J. R. Banavar, S. P. Hubbell, and A. Maritan, "Patterns of relative species abundance in rainforests and coral reefs," *Nature,* vol. 450, pp. 45-49, 2007. [9] C. J. Chu, Y. S. Wang, G. Z. Du, F. T. Maestre, Y. J. Luo, and G. Wang, "On the balance

[10] P. B. Adler, "Neutral models fail to reproduce observed species-area and species-time relationships in Kansas grasslands," *Ecology,* vol. 85, pp. 1265-1272, 2004. [11] C. K. Kelly, M. G. Bowler, O. Pybus, and P. H. Harvey, "Phylogeny, niches, and relative abundance in natural communities," *Ecology,* vol. 89, pp. 962-970, 2008. [12] T. Wiegand, S. Gunatilleke, and N. Gunatilleke, "Species associations in a heterogeneous Sri lankan dipterocarp forest," *American Naturalist,* vol. 170, pp. E67-E95, 2007. [13] E. G. Leigh, "Neutral theory: a historical perspective," *Journal of Evolutionary Biology,*

[14] D. Tilman, "Niche tradeoffs, neutrality, and community structure: A stochastic theory

[15] P. Amarasekare, "The geometry of coexistence," *Biological Journal of the Linnean Society,*

[16] P. Chesson and J. J. Kuang, "The interaction between predation and competition"

[17] D. Tilman, *Resource Competition and Community Structure*. Princeton, New Jersey:

[18] P. Amarasekare, M. F. Hoopes, N. Mouquet, and M. Holyoak, "Mechanisms of

[19] P. Chesson, R. L. E. Gebauer, S. Schwinning, N. Huntly, K. Wiegand, M. S. K. Ernest,

[20] P. Chesson, "Quantifying and testing species coexistence mechanisms," in *Unity in*

coexistence in competitive metacommunities," *The American Naturalist,* vol. 164, 2004.

A. Sher, A. Novoplansky, and J. F. Weltzin, "Resource pulses, species interactions and diversity maintenance in arid and semi-arid environments," *Oecologia,* vol. 141,

*Diversity: Reflections on Ecology after the Legacy of Ramon Margalef*, F. Valladares, A.

of resource competition, invasion, and community assembly," *Proceedings of the*

between niche and neutral processes as drivers of community structure along a successional gradient: Insights from alpine and sub-alpine meadow communities,"

*approaches*. Chicago: The University of Chicago Press, 2003.

*Theory*, J. Roughgarden, R. M. May, and S. A. Levin, Eds. Princeton, N.J.: Princeton

**6. References**

Press, 1995.

University Press, 1989, pp. 203-226.

*and Systematics,* vol. 31, pp. 343–66, 2000.

*Annals of Botany,* vol. 100, pp. 807-812, 2007.

Princeton University Press, 2001.

vol. 20, pp. 2075-2091, 2007.

vol. 71, pp. 1-31, 2000.

pp. 236-257, 2004.

*National Academy of Science USA,* 2004.

*Nature*, Vol 456, pp 235-238, 2008.

Princeton University Press, 1982.

Camacho, A. Elosegui, C. Gracia, M. Estrada, J. C. Senar, and J. M. Gili, Eds. Bilbao: Fundacion BBVA, 2008, pp. 119-164.

[61] M. Huston, "A general hypothesis of species diversity," *The American Naturalist,* vol.

[62] P. Amarasekare and R. M. Nisbet, "Spatial heterogeneity, source-sink dynamics, and

[63] P. L. Chesson, "Coexistence of competitors in spatially and temporally varying

[64] P. Chesson, "General theory of competitive coexistence in spatially-varying environments," *Theoretical Population Biology,* vol. 58, pp. 211-237, 2000. [65] R. E. Snyder, "Spatiotemporal population distributions and their implications for

[66] A. L. W. Sears, "Quantifying the effects of spatial environmental variation on dynamics

[67] D. Tilman, *Plant Strategies and the Dynamics and Structure of Plant Communities*.

[68] R. E. Snyder, E. T. Borer, and P. Chesson, "Examining the relative importance of spatial and nonspatial coexistence mechanisms," *American Naturalist,* vol. 166, pp. E75-E94, 2005. [69] N. Shigesada, "Spatial distribution of rapidly dispersing animals in heterogeneous

[70] A. S. MacDougall, B. Gilbert, and J. M. Levine, "Plant invasions and the niche," *Journal*

[71] R. M. Keane and M. J. Crawley, "Exotic plant invasions and the enemy release

[72] K. Petren and T. Case, "An experimental demonstration of exploitation competition in

[73] R. M. Callaway and E. T. Aschehout, "Invasive plants versus their new and old neighbors: a mechanism for exotic invasion," *Science,* vol. 290, pp. 521-523, 2000. [74] A. A. Sher and L. A. Hyatt, "The disturbed resource-flux invasion matrix: a new framework for patterns of plant invasion," *Biological Invasions,* vol. 1, pp. 107-114, 1999. [75] C. M. Clark, E. E. Cleland, S. L. Collins, J. E. Fargione, L. Gough, K. L. Gross, S. C.

[76] M. L. Brooks, "Effects of increased soil nitrogen on the dominance of alien annual plants in the Mojave Desert," *Journal of Applied Ecology,* vol. 40, pp. 344-353, 2003. [77] D. A. Wedin and D. Tilman, "Influence of nitrogen loading and species composition on the carbon balance of grasslands," *Science,* vol. 274, pp. 1720-1723, 1996. [78] J. R. Malcolm, C. R. Liu, R. P. Neilson, L. Hansen, and L. Hannah, "Global warming

[79] S. Vuilleumier, C. Wilcox, B. J. Cairns, and H. P. Possingham, "How patch

[80] J. H. Connell, M. D. Lowman, and I. R. Noble, "Subcanopy gaps in temperate and tropical forests," *Austalian Journal of Ecology,* vol. 22, pp. 163-168, 1997.

Pennings, K. N. Suding, and J. B. Grace, "Environmental and plant community determinants of species loss following nitrogen enrichment," *Ecology Letters,* vol. 10,

and extinctions of endemic species from biodiversity hotspots," *Conservation*

configuration affects the impact of disturbances on metapopulation persistence,"

*Theoretical Population Biology,* vol. 28, pp. 263-287, 1985.

Princeton, New Jersey: Princeton University Press, 1988.

environments.," *Lect. Notes Biomath.,* vol. 54, pp. 478-501., 1984.

hypothesis," *Trends in Ecology and Evolution,* p. this issue, 2002.

an ongoing invasion," *Ecology,* vol. 77, pp. 118-132, 1996.

the local coexistence of competing species," *The American Naturalist,* vol. 158, pp.

environments: a look at the combined effects of different sorts of variability,"

species coexistence in a variable environment," *Theoretical Population Biology,* vol.

of natural plant populations: field tests for covariance between response to the environment and competition," in *Population biology* Davis: University of California,

113, pp. 81-101, 1979.

572-584, 2001.

72, pp. 7-20, 2007.

Davis, 2004, 155 pp.

pp. 596-607, 2007.

*of Ecology,* vol. 97, pp. 609-615, 2009.

*Biology,* vol. 20, pp. 538-548, 2006.

*Theoretical Population Biology,* vol. 72, pp. 77-85, 2007.

[42] J. P. Grover and R. D. Holt, "Disentangling resource and apparent competition: realistic

[43] D. W. Stephens and J. R. Krebs, *Foraging Theory*. Princeton, N. J.: The Princeton

[44] D. R. Strong, "Density vagueness: abiding the variance in the demography of real

[45] Y. Iwasa and J. Roughgarden, "Interspecific competition among metapopulations with space-limited subpopulations," *Theoretical Population Biology,* vol. 30, pp. 194-214, 1986. [46] D. Tilman, "Competition and biodiversity in spatially structured habitats," *Ecology,* vol.

[47] R. E. Snyder and P. Chesson, "How the spatial scales of dispersal, competition, and

[48] B. M. Bolker, S. W. Pacala, and C. Neuhauser, "Spatial dynamics in model plant

[49] P. Amarasekare, "Competitive coexistence in spatially structured environments: a

[50] S. Muko and Y. Iwasa, "Incomplete mixing promotes species coexistence in a lottery

[51] J. J. Kuang and P. Chesson, "Interacting coexistence mechanisms in annual plant

[52] P. Chesson and J. J. Kuang, "The storage effect due to frequency-dependent predation

[53] P. B. Adler, S. P. Ellner, and J. M. Levine, "Coexistence of perennial plants: an embarrassment of niches," *Ecology Letters,* vol. 13, pp. 1019-1029, 2010. [54] P. B. Adler, J. HilleRisLambers, P. C. Kyriakidis, Q. F. Guan, and J. M. Levine, "Climate

[55] A. L. Angert, T. E. Huxman, P. Chesson, and D. L. Venable, "Functional tradeoffs

[57] J. H. Connell, "Diversity and the coevolution of competitors, or the ghost of

[58] G. E. Hutchinson, "The paradox of the plankton," *The American Naturalist,* vol. 95, pp.

[59] J. H. Connell, "Diversity in tropical rain forests and coral reefs," *Science,* vol. 199, pp.

[60] P. L. Chesson and T. J. Case, "Overview: nonequilibrium community theories: chance,

variability, history, and coexistence," in *Community Ecology*, J. Diamond and T. J.

synthesis," *Ecology Letters,* vol. 6, pp. 1109-1122, 2003.

*Population Biology,* vol. 77, pp. 56-70, 2010.

*Animal Ecology,* vol. 69, pp. 201-213, 2000.

competition past," *Oikos,* vol. 35, pp. 131-138, 1980.

Case, Eds. New York: Harper and Row, 1986, pp. 229-239.

353-376, 1998.

University Press, 1986.

75, pp. 2-16, 1994.

pp. 359-368, 2003.

164, 2010.

12798, 2006.

137-145, 1961.

1302-1310, 1978.

2003.

164, pp. 633-650, 2004.

Harper & Row Publishing, 1986.

models for plant-herbivore communities," *Journal of Theoretical biology,* vol. 191, pp.

populations.," in *Community Ecology*, J. Diamond and T. e. Case, Eds. New York:

environmental heterogeneity interact to affect coexistence," *American Naturalist,* vol.

communities: What do we really know?," *American Naturalist,* vol. 162, pp. 135-148,

model with permanent spatial heterogeneity," *Theoretical Population Biology,* vol. 64,

communities: frequency-dependent predation and the storage effect " *Theoretical*

in multispecies plant communities," *Theoretical Population Biology,* vol. 78, pp. 148-

variability has a stabilizing effect on the coexistence of prairie grasses," *Proceedings of the National Academy of Sciences of the United States of America,* vol. 103, pp. 12793-

determine species coexistence via the storage effect," *Proceedings of the National Academy of Sciences of the United States of America,* vol. 106, pp. 11641-11645, 2009. [56] P. Amarasekare, "Spatial dynamics in a host-multiparasitoid community," *Journal of*

**4**

*Hungary*

**Using Multiple Linear Regression**

**Avian Species Imperilment in**

R. Eliot Crafton and Brandon P. Anthony *Department of Environmental Sciences & Policy,*

 *Central European University, Budapest*

**Sub-Saharan Africa and Europe**

**Models to Identify Factors Underlying**

Determining what factors influence the threats faced by the world's flora and fauna is of key importance to conservation biologists (Cardillo et al., 2008; Davies et al., 2006; Smith et al., 2003; see Spangenberg, 2002). A plethora of research has been directed at this effort and has looked extensively at biological and anthropogenic factors, including social and socioeconomic conditions (e.g. Holland et al., 2009; Huby et al., 2006; Kerr & Currie, 1995; Lenzen et al., 2009; McKee et al., 2003). This chapter intends to supplement the existing literature by utilizing updated data to address this issue from a primarily socio-economic perspective for birds in a selection of sub-Saharan African and European countries. We generate several models using multiple linear regression to test the explanatory power of a host of variables, including human population density (HPD) per km2, Corruption Perception Index (CPI) score (as a proxy for governance), GDP per capita, and the average degrees from the equator. In addition, the results are considered in light of projected changes to HPD levels

Bird species are currently being impacted by several threats, resulting in the need for conservationists to address a wide range of issues (Brooke et al., 2008).  These threats have the potential to impact the quality or quantity of available resources, directly impact the population, or change the conditions that a species may face. In addition, while these threats exist in isolation, many are correlated or exist in a cascading fashion (for example climate change can lead to habitat destruction). While these threats, and others, can be enumerated, the underlying drivers of these pressures are often rooted in socio-economic conditions, including corruption, HPD, and poverty level (Kerr & Currie, 1995; Pandit & Laband, 2009;

Understanding how these socioeconomic factors influence conservation agendas has been the focus of several authors, occasionally with contradictory results. While correlation can

**1. Introduction**

for the year 2050 (United Nations [UN], 2008).

Pellegrini & Gerlagh, 2004).


**Abstract**

Flavonoids are a major class of secondary metabolites that comprises more than 6000 compounds that have been identified. They are biosynthesized via the phenylpropanoid metabolic pathway that involves groups of enzymes such as isomerases, hydroxylases, and reductases that greatly affect the determination of the flavonoid skeleton. For example, transferase enzymes responsible for the modification of sugar result in changes in the physiological activity of the flavonoids and changes in their physical properties, such as solubility, reactivity, and interaction with cellular target molecules, which affect their pharmacodynamics and pharmacokinetic properties. In addition, flavonoids have diverse biological activities such as antioxidants, anticancer, and antiviral in managing Alzheimer's disease. However, most marine flavonoids are still incompletely discovered because marine flavonoid biosynthesis is produced and possesses unique substitutions that are not commonly found in terrestrial bioactive compounds. The current chapter will illustrate the importance of flavonoids' role in metabolism and the main difference between marine and terrestrial flavonoids.

**Keywords:** marine flavonoids, biosynthesis, pharmacodynamics, pharmacokinetics, defense mechanism


2.4.2. Test Factors and Levels

The test factors were the speed *n* of the toothed roller, ground clearance *α*, and cogging angle *θ*. According to the results of calculations, the speed of the V-shaped toothed roller varied between 176 and 500 r/min; thus, the test speeds were preliminarily determined to be 200, 300, and 400 r/min. The results of the theoretical analysis indicated that operating the toothed roller at a higher ground clearance yields better performance; thus, *α* was assigned values of −20 mm (i.e., 20 mm into the soil), 20 mm, and 60 mm in this study. Based on the optimized above-described structural design, the cogging angle *θ* should range between 25◦ and 38.6◦. However, to account for the coefficient of friction between the cotton stalk and steel plate and to minimize processing difficulty, the maximum cogging angle was set to 40◦ for the field test. In the case of a certain thrust, the smaller the cogging angle, the greater the clamping force on the cotton stalk. However, there are two uncertainties: 1. Too small an angle leads to too long tooth height, which easily causes the insufficient strength of tooth plate structure; 2. Excessive clamping force will easily cause the cotton stalks to fall off easily. Thus, the cogging angles implemented in the field test were 25◦, 32.5◦, and 40◦. The test levels are summarized in Table 2. The L9 (34) orthogonal

experiment was designed to have nine test groups. The experiment was repeated three times for each group, and the mean value was calculated.

**Table 2.** Factors and levels of orthogonal experiment.

#### 2.4.3. Test Methods

The test was carried out in accordance with the standard GB/T8097-2008 [26]. To ensure stable operation of the equipment and minimize error, the puller machine was allowed to adjust its operating posture by traveling a distance of 20 m before data were collected. To facilitate the process of data collection, two rows of cotton stalks were harvested each time. The data collection region was 30 m long, corresponding to approximately 200 cotton stalks. The evaluation index for the test was the removal rate; it was determined as follows:

$$y = \frac{M\_b}{M\_z} \times 100\% \tag{16}$$

In the formula, *y*—the removal rate of cotton stalks, %; *Mb*—the number of cotton stalks pulled out, plant;

*Mz*—the sum of cotton stalks, plants.

#### **3. Results and Discussion**

The test plan and results were shown in Table 3 (A, B, and C are the ground clearance, the rotation speed, and the cogging angle, respectively).

**Table 3.** Test results and analysis.

According to the results of numerical analysis for the range *R* for each factor in Table 2, it can be seen that the order of significance in terms of the influence of each factor on the evaluation index (i.e., the removal rate) was *A* > *B* > *C*. The ground clearance most significantly influenced the removal rate, followed by the rotational speed, and the cogging angle. To optimize the combination of these factors to obtain the highest cotton stalk removal rate, the removal rate was plotted as a function of the averaged test factor results presented in Table 2; the resulting graph is shown in Figure 14. It can be ascertained from

Figure 12 that the combination of factors that yielded the highest removal rate was *A*1*B*2*C*1, i.e., the combination of a ground clearance of −20 mm, a rotational speed of 300 r/min, and cogging angle of 25◦.

**Figure 14.** Relationship between factor level and removal rate.

Minitab software was used as a platform to analyze the variance of the results of the orthogonal experiment and quantify the respective influences of the three factors (i.e., cogging angle, rotational speed, and ground clearance) on the removal rate; the results are summarized in Table 4.

**Table 4.** Analysis of variance for orthogonal experiment results.

Note: *p* < 0.01 (highly significant, \*\*); *p* < 0.05 (significant, \*). Based on the analysis of variance results presented in Table 3, it can be ascertained on the basis of FA > FB > FC that A had an extremely significant influence on the removal rate, B had a significant influence, and C had minimal influence. Such results are consistent with the range analysis results. In addition, it can be also ascertained from Table 3 that the sum of squares error was much less than that of factor A, factor B, or factor C, indicating that the correlations between test factors did not considerably affect the evaluation index.

Compared with the knife-roller type stalk pulling operation equipment, it has the advantages of not entering the soil and reducing energy consumption on the basis of nonalignment operation. Tests have proved that in the Binzhou area, cotton stalks with larger diameters can be effectively pulled out. However, since this type of stalk-pulling device has many V-shaped structures on its stalk-pulling rollers, once cotton stalks are entangled, the cotton stalks will be damaged. The damaged cotton stalk cannot fall off, which leads to its working failure; secondly, this device has been tested in a densely planted cotton area. When the average root diameter of cotton stalks is about 10 mm, the stalk-pulling effect is very poor, so this device still needs further in-depth research. The most foreign equipment introduced in the literature is the double-roller type stalk pulling equipment. Our team has used the domestically produced double-roller equipment to conduct experiments in the Binzhou area, and the effect is not ideal. The variety and planting mode of cotton stalks have a great influence on the mechanical properties of cotton stalks. Up to now, there is no stalk-pulling equipment that can adapt to cotton stalks in different regions. Therefore, further research is needed to solve the issue of stalk-pulling technology.


**5. Novel strategies to overcome TRAIL resistance**

During the last decades, a better understanding of cancer biology has led to the development of new promising therapeutic approaches, based on "molecular targeted" drugs, directed against specific "target" molecules playing a key role in tumour maintenance (Urruticoechea et al., 2010). Based on the principle that inhibiting as many targets as possible reduces the emergence of drug resistance, the use of combined therapies or multi-target inhibitors is gaining field in the design of new treatments. A number of chemical and physical anticancer strategies have been developed to bypass TRAIL resistance, based on the combination of rTRAIL or agonistic antibodies with chemotherapeutic agents, irradiation, or targeted small molecules, like proteasome, histone deacetylase or NF-B inhibitors (Testa, 2010; Russo et al., 2010). The agents used in combination with TRAIL either enhance TRAIL-R1/-R2 expression or decrease expression of anti-apoptotic proteins (c-FLIP, X-IAP, Bcl-2) (Mellier et al., 2010) (Fig. 2). Many of these combinatorial therapies hold promise for future developments in the treatment of haematological malignancies since they may reduce excessive systemic toxicity toward normal cells and resistance of tumour cells after recurrent treatments. We and other authors have demonstrated that TRAIL-mediated cytotoxicity is increased by ionizing radiation and chemotherapy in both myeloid and erythroid leukaemia cell lines as well as in T lymphoma cell lines (Gong & Almasan, 2000; Di Pietro et al., 2001; Sabatini et al., 2004; Zauli et al., 2005; Caravatta et al., 2008; Impicciatore et al., 2010; Signore et al., 2011). Although an increasing number of drugs warrant further investigation as potential new strategies for the treatment of solid tumours or AML in combination with soluble rTRAIL (Suh et al., 2003), only in few cases the efficacy of the combined treatments has been proved *in vivo* and a general consensus on how chemotherapy and radiotherapy may synergize with TRAIL therapy is far to be reached (Russo et al., 2010).

#### **5.1 TRAIL-death receptor-targeted treatment**

A number of receptor-specific TRAIL-variants and agonistic antibodies have been recently developed. Some of these soluble rTRAIL and MoAbs targeting TRAIL-R1 and/or TRAIL-R2 (TRAIL receptor agonists, TRAs) are progressing to phase I/II clinical trials (Mahmood &

clones. Interestingly, also thalidomide, which holds great promise as a new anti-neoplastic agent for the treatment of refractory MM, triggers activation of caspase-8 and downregulates NF-B activity and c-FLIP (Mitsiades et al, 2002). These studies form the basis for clinical trials of these agents, alone and coupled with conventional and novel therapies, to improve outcome in MM. It is worth underlining that while the potential therapeutic use of rTRAIL or TRAs in most myeloid and lymphoid malignancies is still to be evaluated, rTRAIL appears to be a very promising candidate for the therapy of MM, either alone or in combination with valproic acid, a histone deacetylase inhibitor, arsenic trioxide, IFN, or with the low-molecular-weight Smac mimetic LBW242 (Secchiero & Zauli, 2008). Moreover, the use of specific anti-TRAIL-R1 or anti-TRAIL-R2 agonistic antibodies, more than the treatment with TRAIL itself (Locklin et al., 2007), has proved an effective strategy to counteract OPG-mediated effects and increase TRAIL-induced apoptosis of MM cells (Secchiero & Zauli, 2008). Of note, other preclinical studies aimed at targeting the RANK/RANKL/OPG pathway have paved the way to clinical experimentation likely to

During the last decades, a better understanding of cancer biology has led to the development of new promising therapeutic approaches, based on "molecular targeted" drugs, directed against specific "target" molecules playing a key role in tumour maintenance (Urruticoechea et al., 2010). Based on the principle that inhibiting as many targets as possible reduces the emergence of drug resistance, the use of combined therapies or multi-target inhibitors is gaining field in the design of new treatments. A number of chemical and physical anticancer strategies have been developed to bypass TRAIL resistance, based on the combination of rTRAIL or agonistic antibodies with chemotherapeutic agents, irradiation, or targeted small molecules, like proteasome, histone deacetylase or NF-B inhibitors (Testa, 2010; Russo et al., 2010). The agents used in combination with TRAIL either enhance TRAIL-R1/-R2 expression or decrease expression of anti-apoptotic proteins (c-FLIP, X-IAP, Bcl-2) (Mellier et al., 2010) (Fig. 2). Many of these combinatorial therapies hold promise for future developments in the treatment of haematological malignancies since they may reduce excessive systemic toxicity toward normal cells and resistance of tumour cells after recurrent treatments. We and other authors have demonstrated that TRAIL-mediated cytotoxicity is increased by ionizing radiation and chemotherapy in both myeloid and erythroid leukaemia cell lines as well as in T lymphoma cell lines (Gong & Almasan, 2000; Di Pietro et al., 2001; Sabatini et al., 2004; Zauli et al., 2005; Caravatta et al., 2008; Impicciatore et al., 2010; Signore et al., 2011). Although an increasing number of drugs warrant further investigation as potential new strategies for the treatment of solid tumours or AML in combination with soluble rTRAIL (Suh et al., 2003), only in few cases the efficacy of the combined treatments has been proved *in vivo* and a general consensus on how chemotherapy and radiotherapy may synergize with TRAIL therapy is

A number of receptor-specific TRAIL-variants and agonistic antibodies have been recently developed. Some of these soluble rTRAIL and MoAbs targeting TRAIL-R1 and/or TRAIL-R2 (TRAIL receptor agonists, TRAs) are progressing to phase I/II clinical trials (Mahmood &

lead to new therapeutic approaches (Buckle et al., 2010).

far to be reached (Russo et al., 2010).

**5.1 TRAIL-death receptor-targeted treatment**

**5. Novel strategies to overcome TRAIL resistance**

Shukla, 2010). The clinical use of TRAs is a very promising and innovative approach to increase selectivity and reduce undesired toxicity of cancer treatments in comparison with modern anticancer drugs (protein kinase inhibitors or MoAb agonists for growth receptors) (Russo et al., 2010). These compounds were generated to selectively bind and activate their respective DRs without affecting decoy receptors or OPG. DRs engagement, using recombinant death ligands or agonistic antibodies, leads to the activation of both extrinsic and intrinsic apoptosis pathways, while, generally, chemotherapy or radiotherapy triggers the mitochondrial/intrinsic pathway (Fig. 2). Therefore, the conventional therapeutic approach could be implemented by DR-induced apoptosis when DRs are expressed and functional on tumour cells. As already mentioned, although soluble rTRAIL as well as TRAs are not completely free from toxicity, both reagents elicit a significant lower hepatotoxicity when administered systemically compared to CD95 receptor agonists (Lawrence et al., 2001). Besides the advantage of an improved specificity and a lower toxicity of TRAs over TRAIL ligand, pharmacokinetic studies performed in primates and humans have shown that these agents have a longer half-life (around 15 days) than soluble TRAIL (30 min) that makes them easier to dose and administer (Duiker et al., 2006). Preclinical studies performed *in vitro* in cultured human cell lines and *in vivo* in murine xenograft cancer models (Cretney et al., 2007) showed favourable results when TRAs were used as single agents and enhanced cytotoxicity when they were combined with chemotherapy or radiotherapy (Marini et al., 2006). In particular, HGS-ETR1 (anti-TRAIL-R1, mapatumumab) as well as HGS-ETR2 (anti-TRAIL-R2, lexatumumab) was able to induce apoptosis in primary and cultured lymphoma cells increasing cell death when associated either with conventional chemotherapy (doxorubicin) or novel drugs like proteasome inhibitors (bortezomib) (Georgakis et al., 2005). As well, multiple solid tumours including lung, colon and renal carcinoma were found responsive to TRAs treatment used alone or in combination with chemotherapy (Pukac et al., 2005). To date, the fully humanized MoAbs HGS-ETR1, HGS-ETR2 and HGS-TR2J (anti-TRAIL-R2) (all three from Human Genome Sciences, Rockville, MD) are used in ongoing trials for the treatment of advanced solid tumours, lymphoma or MM (Mahmood & Shukla, 2010). A number of excellent reviews on different therapeutic approaches to specifically target TRAIL and DR pathways have been recently published (Ashkenazi et al., 2008; Papenfuss et al., 2008; Mahmood & Shukla, 2010; Russo et al., 2010).

Of particular interest is the current use of rTRAIL and TRAs for the treatment of B cell malignancies (Mahmood & Shuka, 2010). As shown by other authors, the DR pathway is intact and functional in various types of cancers, including B-cell lymphomas (Snell et al., 1997; Georgakis et al., 2005). B-cell sensitivity to TRAs is a fundamental requirement for therapeutic efficacy, since TRAIL-R1 and TRAIL–R2 mutations, observed in NHL as well as in other human tumours (Lee et al., 2001), make neoplastic B cells insensitive to TRAIL and, presumably, to agonistic antibodies mimicking its action. TRAIL-R1 and TRAIL-R2 map to human chromosome 8p21-22, a site of frequent allelic loss in tumours. This led to the hypothesis that, as potential tumour suppressors, TRAIL-Rs may also harbour somatic mutations in human tumours. The most frequent mutations identified so far concern TRAIL-R2 and affect the intracellular domain of the receptor, i.e. the FADD-binding domain, and, as a consequence, its capability of inducing apoptosis (Bin et al., 2007). Although still poor is the knowledge of how TRAIL-Rs mutations affect signalling events, it is predictable that a patient displaying a TRAIL-R2 mutation would not benefit from treatment with either rTRAIL or an anti-TRAIL-R2 antibody but from treatment with mapatumumab or a

Signalling Pathways Leading to TRAIL Resistance 215

Besides oncogenes over-expression and cell cycle control mechanisms disruption, mutations in apoptotic regulators (namely p53) are very frequent in cancer cells and represent for them a way to escape toxic effects inducible with radio-chemotherapy. As an alternative strategy to restoring transcriptional activation to mutant p53 proteins in solid tumours, small molecule selective inhibitors of p53/MDM2 interaction (Nutlins) are emerging as an innovative tool in the treatment of malignancies expressing wtp53 including haematological disorders (Secchiero et al., 2008; Impicciatore et al., 2010). Nutlins were the first potent and selective small molecules, antagonists of the p53/MDM2 interaction, to be identified 7 years ago (Vassilev et al., 2004). Since then several classes of small-molecule inhibitors with distinct chemical structure have been reported (Shangary & Wang, 2009), although only Nutlin-3 has been extensively evaluated for its therapeutic potential and mechanism of action in human cancer and represents a promising therapeutic candidate for drug development (Shangary & Wang, 2009). Several authors have investigated the effects of Nutlins, used alone or in combination with other therapeutic agents, on primary cells, different cell lines and tumour xenografts (Kojima et al., 2005; Lehmann et al., 2007). In particular, it has been reported that the active enantiomer Nutlin-3a induces i) increased levels of p53, ii) p53- and p21-dependent cell cycle arrest and iii) p53-dependent apoptosis in a number of solid tumours and haematological malignancies including primary AML (Kojima et al., 2005), MM (Stuhmer et al., 2005), B-CLL (Coll-Mulet et al., 2006) and Hodgkin lymphomas (HL) (Drakos et al., 2007). Unlike radiation and conventional chemotherapy, MDM2 inhibitors induce accumulation and activation of p53 in cancer and normal cells without inducing DNA damage or post-translational modifications of p53. Nutlins in fact restore p53 function in wtp53 tumour cells without inducing p53 phosphorylation and with limited effects on primary cells (Vassilev et al., 2004). Interestingly, when used at concentrations higher than 10 mM, Nutlin-3, MI-63 and MI-219 are able to inhibit cell proliferation even in cancer cells lacking wtp53 (Shangary & Wang, 2009). In response to Nutlin-3 treatment TRAIL-R2 is up-regulated in B-CLL cells in a p53-dependent manner (Coll-Mulet et al., 2006). Moreover, Nutlins reduce the MDM2 ability to stimulate p53 degradation and represent a promising approach for improving radiotherapy effects especially for tumours over-expressing MDM2 such as sarcomas, solid tumours (Momand et

Besides the use of a broad range of protein inhibitors, chemotherapeutic agents or irradiation to exert synergistic effects with TRAIL action (Mahalingam et al., 2009), more recently the use of natural compounds, including polyphenols, has gained increasing interest due to their relative safety and anti-tumour efficacy in preclinical models (Jacquemin et al., 2010). Actually, it has been demonstrated that a number of natural compounds are able to enhance TRAIL-induced apoptosis in leukaemia cells (Fas et al., 2006; Russo et al., 2007; Hussain et al., 2008; Sung et al. 2010). In particular, it has been shown that wogonin, derived from a popular Chinese herb, attenuates NF-B activity (Fas et al., 2006), whereas curcumin, responsible for the yellow colour of the spice turmeric, upregulates TRAIL-R2 expression and inactivates NF-B in a ROS-dependent manner in a number of solid tumours including Burkitt's lymphoma (Hussain et al., 2008). Moreover, Russo et al. (2007) reported that leukaemia cell lines were efficiently sensitized by quercetin and TRAIL co-treatment through the inhibition of the Akt pathway, while Sung et al., 2010 observed other survival proteins down-regulation after TRAIL and triterpenoids co-

**5.3 Small molecules and natural compounds**

al., 1998) and NHL (Finnegan et al., 1994).

modified version of rTRAIL able to target only one death receptor (MacFarlane et al., 2005). Similarly to TRAIL, TRAs (mapatumumab and lexatumumab) are capable of inducing antilymphoma effects both *in vitro* and *in vivo* (Motoki et al., 2005). In particular, it has been recently demonstrated that mapatumumab can trigger apoptosis through caspase-8 activation via the extrinsic apoptotic pathway (Maddiplata et al., 2007). Various groups of investigators have shown that the activation of the TRAIL-Rs by either ligands or MoAbs sensitizes cancer cells to the effects of various chemotherapeutic and/or biological agents (Secchiero et al., 2007), although in a recent report no correlation between the degree of antitumour *in vitro* activity of mapatumumab and TRAIL-R1 antigen density has been shown (Maddiplata et al., 2007). To explain their results these investigators hypothesized differences in the receptor hetero-dimerization between various B-cell lymphoma cells upon TRAIL-R1 binding to mapatumumab. In fact, it has been published that following *in vitro* exposure to TRAIL ligand or TRAIL-R1 agonists, other DRs are recruited via trimerization, leading to signal transduction and apoptosis (Cretney et al., 2007). Depending on the type of DR undergoing trimerization upon TRAIL-R1 binding the intensity and type of response could change. In theory, the same principle could justify the absence of anti-tumour activity of lexatumumab despite ample surface expression of TRAIL-R2 in the cell lines tested *in vitro* (Maddiplata et al., 2007). Further studies are needed to address this issue.

#### **5.2 Proteasome and histone deacetylase inhibitors**

Inhibition of NF-B (e.g. with mutant forms of IB or proteasome inhibitors) has also been shown to increase TRAIL responsiveness (Sayers & Murphy, 2006). In this respect, besides the clinical use of bortezomib (Velcade, PS-341) for the treatment of multiple myeloma (Sayers & Murphy, 2006), it has been recently reported that treatment with the proteasome inhibitors MG-132 and PS-341 is associated with the up-regulation of TRAIL and its death receptors, TRAIL-R1/TRAIL-R2, in primary B-CLL cells and in the Burkitt lymphoma cell line, BJAB (Kabore et al., 2006). Interestingly, the combined treatment with TRAIL or TRAs and proteasome inhibitors leads to a significant apoptosis induction in B-CLL but not in normal B cells (Kabore et al., 2006). DRs up-regulation by PS-341 was attributed to TRAIL-R2 mRNA stabilization and the consequent increased receptor half-life (Kamdasamy & Kraft, 2008). In addition to the proteasome inhibitors, inhibition of histone deacetylase (HDAC) class I sensitizes B-CLL to TRAIL-induced apoptosis (Hamilton et al., 2010). An aberrant regulation of gene expression due to alterations in histone acetyltransferase (HAT) or HDAC recruitment and activity has been constantly found in both solid and haematological tumours (Mai & Altucci, 2009). Therefore HDAC can be considered as potential therapeutic targets of human malignancies. Interestingly, the reduction of TRAIL protein degradation has been recently observed in thyroid cancer cells and proposed as a novel action of HDAC inhibitors (Borbone et al., 2010). It is worth outlining that HDAC inhibitors exert anti-tumour effects at doses that are well tolerated by the patients. Hydroxamic acids, such as SAHA (Vorinostat, Zolinza), were recently approved by the US FDA for the treatment of cutaneous manifestations in patients affected with advanced refractory CTCL. In fact, similarly to what has been seen in B-CLL, CTCL cell lines show pronounced resistance to TRAIL cytotoxicity (Braun et al., 2007). Lastly, a phase I study, currently recruiting participants, will use vorinostat in combination with cytarabine and etoposide for the treatment of patients with relapsed and/or refractory acute leukaemia, MDS or myeloproliferative disorders (see for details http://clinicaltrials.gov).

modified version of rTRAIL able to target only one death receptor (MacFarlane et al., 2005). Similarly to TRAIL, TRAs (mapatumumab and lexatumumab) are capable of inducing antilymphoma effects both *in vitro* and *in vivo* (Motoki et al., 2005). In particular, it has been recently demonstrated that mapatumumab can trigger apoptosis through caspase-8 activation via the extrinsic apoptotic pathway (Maddiplata et al., 2007). Various groups of investigators have shown that the activation of the TRAIL-Rs by either ligands or MoAbs sensitizes cancer cells to the effects of various chemotherapeutic and/or biological agents (Secchiero et al., 2007), although in a recent report no correlation between the degree of antitumour *in vitro* activity of mapatumumab and TRAIL-R1 antigen density has been shown (Maddiplata et al., 2007). To explain their results these investigators hypothesized differences in the receptor hetero-dimerization between various B-cell lymphoma cells upon TRAIL-R1 binding to mapatumumab. In fact, it has been published that following *in vitro* exposure to TRAIL ligand or TRAIL-R1 agonists, other DRs are recruited via trimerization, leading to signal transduction and apoptosis (Cretney et al., 2007). Depending on the type of DR undergoing trimerization upon TRAIL-R1 binding the intensity and type of response could change. In theory, the same principle could justify the absence of anti-tumour activity of lexatumumab despite ample surface expression of TRAIL-R2 in the cell lines tested *in*

*vitro* (Maddiplata et al., 2007). Further studies are needed to address this issue.

MDS or myeloproliferative disorders (see for details http://clinicaltrials.gov).

Inhibition of NF-B (e.g. with mutant forms of IB or proteasome inhibitors) has also been shown to increase TRAIL responsiveness (Sayers & Murphy, 2006). In this respect, besides the clinical use of bortezomib (Velcade, PS-341) for the treatment of multiple myeloma (Sayers & Murphy, 2006), it has been recently reported that treatment with the proteasome inhibitors MG-132 and PS-341 is associated with the up-regulation of TRAIL and its death receptors, TRAIL-R1/TRAIL-R2, in primary B-CLL cells and in the Burkitt lymphoma cell line, BJAB (Kabore et al., 2006). Interestingly, the combined treatment with TRAIL or TRAs and proteasome inhibitors leads to a significant apoptosis induction in B-CLL but not in normal B cells (Kabore et al., 2006). DRs up-regulation by PS-341 was attributed to TRAIL-R2 mRNA stabilization and the consequent increased receptor half-life (Kamdasamy & Kraft, 2008). In addition to the proteasome inhibitors, inhibition of histone deacetylase (HDAC) class I sensitizes B-CLL to TRAIL-induced apoptosis (Hamilton et al., 2010). An aberrant regulation of gene expression due to alterations in histone acetyltransferase (HAT) or HDAC recruitment and activity has been constantly found in both solid and haematological tumours (Mai & Altucci, 2009). Therefore HDAC can be considered as potential therapeutic targets of human malignancies. Interestingly, the reduction of TRAIL protein degradation has been recently observed in thyroid cancer cells and proposed as a novel action of HDAC inhibitors (Borbone et al., 2010). It is worth outlining that HDAC inhibitors exert anti-tumour effects at doses that are well tolerated by the patients. Hydroxamic acids, such as SAHA (Vorinostat, Zolinza), were recently approved by the US FDA for the treatment of cutaneous manifestations in patients affected with advanced refractory CTCL. In fact, similarly to what has been seen in B-CLL, CTCL cell lines show pronounced resistance to TRAIL cytotoxicity (Braun et al., 2007). Lastly, a phase I study, currently recruiting participants, will use vorinostat in combination with cytarabine and etoposide for the treatment of patients with relapsed and/or refractory acute leukaemia,

**5.2 Proteasome and histone deacetylase inhibitors**

#### **5.3 Small molecules and natural compounds**

Besides oncogenes over-expression and cell cycle control mechanisms disruption, mutations in apoptotic regulators (namely p53) are very frequent in cancer cells and represent for them a way to escape toxic effects inducible with radio-chemotherapy. As an alternative strategy to restoring transcriptional activation to mutant p53 proteins in solid tumours, small molecule selective inhibitors of p53/MDM2 interaction (Nutlins) are emerging as an innovative tool in the treatment of malignancies expressing wtp53 including haematological disorders (Secchiero et al., 2008; Impicciatore et al., 2010). Nutlins were the first potent and selective small molecules, antagonists of the p53/MDM2 interaction, to be identified 7 years ago (Vassilev et al., 2004). Since then several classes of small-molecule inhibitors with distinct chemical structure have been reported (Shangary & Wang, 2009), although only Nutlin-3 has been extensively evaluated for its therapeutic potential and mechanism of action in human cancer and represents a promising therapeutic candidate for drug development (Shangary & Wang, 2009). Several authors have investigated the effects of Nutlins, used alone or in combination with other therapeutic agents, on primary cells, different cell lines and tumour xenografts (Kojima et al., 2005; Lehmann et al., 2007). In particular, it has been reported that the active enantiomer Nutlin-3a induces i) increased levels of p53, ii) p53- and p21-dependent cell cycle arrest and iii) p53-dependent apoptosis in a number of solid tumours and haematological malignancies including primary AML (Kojima et al., 2005), MM (Stuhmer et al., 2005), B-CLL (Coll-Mulet et al., 2006) and Hodgkin lymphomas (HL) (Drakos et al., 2007). Unlike radiation and conventional chemotherapy, MDM2 inhibitors induce accumulation and activation of p53 in cancer and normal cells without inducing DNA damage or post-translational modifications of p53. Nutlins in fact restore p53 function in wtp53 tumour cells without inducing p53 phosphorylation and with limited effects on primary cells (Vassilev et al., 2004). Interestingly, when used at concentrations higher than 10 mM, Nutlin-3, MI-63 and MI-219 are able to inhibit cell proliferation even in cancer cells lacking wtp53 (Shangary & Wang, 2009). In response to Nutlin-3 treatment TRAIL-R2 is up-regulated in B-CLL cells in a p53-dependent manner (Coll-Mulet et al., 2006). Moreover, Nutlins reduce the MDM2 ability to stimulate p53 degradation and represent a promising approach for improving radiotherapy effects especially for tumours over-expressing MDM2 such as sarcomas, solid tumours (Momand et al., 1998) and NHL (Finnegan et al., 1994).

Besides the use of a broad range of protein inhibitors, chemotherapeutic agents or irradiation to exert synergistic effects with TRAIL action (Mahalingam et al., 2009), more recently the use of natural compounds, including polyphenols, has gained increasing interest due to their relative safety and anti-tumour efficacy in preclinical models (Jacquemin et al., 2010). Actually, it has been demonstrated that a number of natural compounds are able to enhance TRAIL-induced apoptosis in leukaemia cells (Fas et al., 2006; Russo et al., 2007; Hussain et al., 2008; Sung et al. 2010). In particular, it has been shown that wogonin, derived from a popular Chinese herb, attenuates NF-B activity (Fas et al., 2006), whereas curcumin, responsible for the yellow colour of the spice turmeric, upregulates TRAIL-R2 expression and inactivates NF-B in a ROS-dependent manner in a number of solid tumours including Burkitt's lymphoma (Hussain et al., 2008). Moreover, Russo et al. (2007) reported that leukaemia cell lines were efficiently sensitized by quercetin and TRAIL co-treatment through the inhibition of the Akt pathway, while Sung et al., 2010 observed other survival proteins down-regulation after TRAIL and triterpenoids co-

Signalling Pathways Leading to TRAIL Resistance 217

The author wishes to thank Dr. Francesca Gambacorta for helpful assistance in the artwork.

Abdulghani, J. & El-Deiry, W.S. (2010). TRAIL receptor signaling and therapeutics, *Expert*

Akahanea, K., Inukaia, T., Zhanga, X., Hirosea, K., Kurodaa, I., et al. (2010). Resistance of T-

Almasan, A. & Ashkenazi, A. (2003). Apo2L/TRAIL: apoptosis signaling, biology, and potential for cancer therapy, *Cytokine Growth Factor Rev* 14(3-4):337-348. Ashkenazi, A. & Dixit, V.M. (1999). Apoptosis control by death and decoy receptors, *Curr*

Ashkenazi, A, Holland, P. & Eckhardt, S.G. (2008). Ligand-based targeting of apoptosis in

Barkett, M. & Gilmore, T.D. (1999). Control of apoptosis by Rel/NF-kappaB transcription

Bin, L., Thorburn, J., Thomas, L.R., Clark, P.E., Humphreysm R. & Thorburn, A. (2007).

Blum, A., Chaperot, L., Molens, J.P., Foissaud, V., Plantaz, D. & Plumas, J. (2006).

Bonavida, B. (2007). Rituximab-induced inhibition of antiapoptotic cell survival pathways:

Borbone, E., Berlingieri, M.T., De Bellis, F., Nebbioso, A., Chiappetta, G. et al., (2010).

Braun, F.K., Fecker, L.F., Schwarz, C., Walden, P., Assaf, C., et al. (2007). Blockade of death

Buckle, C.H., Neville-Webbe, H.L., Croucher, P.I. & Lawson, M.A. (2010). Targeting

and novel therapeutic interventions, *Oncogene* 26(25): 3629-3636.

inducing ligand-mediated apoptosis, *Exp Hematol* 38:885–895.

Baeuerle, P.A. & Baltimore, D. (1996). NF-kappa B: ten years after, *Cell* 87(1):13-20.

cell acute lymphoblastic leukemia to tumor necrosis factor-related apoptosis-

cancer: the potential of recombinant human apoptosis ligand 2/tumor necrosis factor–related apoptosis-inducing ligand (rhApo2L/TRAIL), *J Clin Oncol* 26:3621–

Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signalling through the DR4 receptor by competing for ligand binding, *J Biol Chem*, 38: 28189-

Mechanisms of TRAIL-induced apoptosis in leukemic plasmacytoid dendritic cells,

implications in chemo/immunoresistance, rituximab unresponsiveness, prognostic

Histone deacetylase inhibitors induce thyroid cancer-specific apoptosis through proteasome-dependent inhibition of TRAIL degradation, *Oncogene* 29:105-116. Bortul, R., Tazzari, P.L., Cappellini, A., Tabellini, G., Billi, A.M., et al. (2003). Constitutively

active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation, *Leukemia*

receptor-mediated pathways early in the signaling cascade coincides with distinct apoptosis resistance in cutaneous T-cell lymphoma cells, *J Invest Dermatol*

RANK/RANKL in the treatment of solid tumours and myeloma, *Curr Pharm Des*

**7. Acknowledgment**

3630.

28194.

17:379–389.

127(10):2425-2437.

16(11):1272-1283.

*Opin Ther Targets* 14(10):1091-1108.

*Opin Cell Biol* 11:255-260.

factors, *Oncogene* 18:6910-6924.

*Exp Hematol* 34:1655–1662.

**8. References**

treatment. Taken together, these findings demonstrate that nongenotoxic natural molecules or small compounds enhance TRAIL-mediated killing of tumour cells with reduced side effects compared to conventional radio-chemotherapy.

Fig. 2. Schematic representation of molecular mechanisms of novel strategies aimed at restoring TRAIL sensitivity of haematological malignancies. RAD: radiotherapy; CHT: chemotherapy. See text for other abbreviations.